Dataset Information

In vitro and pilot in vivo imaging of 18 kDa translocator protein (TSPO) in inflammatory vascular disease.

ABSTRACT:

Background

Inflammatory vascular disease of the arteries, such as inflamed atheromatous plaques or arteritis, may cause aneurysms or ischemic strokes. In this context, using positron emission tomography (PET) to image inflammation may help select patients who would benefit from appropriate therapeutic interventions. This study sought to assess the usefulness of the 18 kDa translocator protein (TSPO) tracers [¹¹C]-PBR28 and [¹⁸F]-PBR06 for imaging inflammatory vascular disease in vitro and in vivo. Immunohistochemistry for macrophage infiltration as well as autoradiography with [¹⁸F]-PBR06 were performed on eight paraffin-embedded, formalin-fixed atherosclerosis plaques prospectively collected after carotid endarterectomy of eight patients affected by ischemic stroke. Six different patients, one of whom was also included in the in vitro study, underwent PET imaging. Two patients with carotid stenosis associated with ischemic stroke were imaged with [¹⁸F]-PBR06 PET/CT, and four other patients (three with large vessel vasculitis and one with bilateral carotid stenosis but without stroke) were imaged with [¹¹C]-PBR28.

Results

All in vitro sections showed specific binding of [¹⁸F]-PBR06, which co-localized with immunohistochemistry markers for inflammation. However, in vivo TSPO imaging with either [¹¹C]-PBR28 or [¹⁸F]-PBR06 was negative in all participants.

Conclusion

Despite good uptake on surgical samples in vitro, [¹¹C]-PBR28 and [¹⁸F]-PBR06 are not viable clinical tools for imaging inflammatory vascular disease.

Trial registration

NCT02513589, registered 31 July 2015 and NCT00547976, registered 23 October 2007. https://clinicaltrials.gov .

SUBMITTER: Schollhammer R

PROVIDER: S-EPMC8099943 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:In the brain neuropil, translocator protein 18 kDa (TSPO) is a stress response protein that is upregulated in microglia and astrocytes in diverse central nervous system pathologies. TSPO is widely used as a biomarker of neuroinflammation in preclinical and clinical neuroimaging studies. However, there is a paucity of knowledge on the function(s) of TSPO in glial cells. In this study, we explored a putative interaction between TSPO and NADPH oxidase 2 (NOX2) in microglia. We found that TSPO associates with gp91phox and p22phox, the principal subunits of NOX2 in primary murine microglia. The association of TSPO with gp91phox and p22phox was observed using co-immunoprecipitation, confocal immunofluorescence imaging, and proximity ligation assay. We found that besides gp91phox and p22phox, voltage-dependent anion channel (VDAC) also co-immunoprecipitated with TSPO consistent with previous reports. When we compared lipopolysaccharide (LPS) stimulated microglia to vehicle control, we found that a lower amount of gp91phox and p22phox protein co-immunoprecipitated with TSPO suggesting a disruption of the TSPO-NOX2 subunits association. TSPO immuno-gold electron microscopy confirmed that TSPO is present in the outer mitochondrial membrane but it is also found in the endoplasmic reticulum (ER), mitochondria-associated ER membrane (MAM), and in the plasma membrane. TSPO localization at the MAM may represent a subcellular site where TSPO interacts with gp91phox and p22phox since the MAM is a point of communication between outer mitochondria membrane proteins (TSPO) and ER proteins (gp91phox and p22phox) where they mature and form the cytochrome b558 (Cytb558) heterodimer. We also found that an acute burst of reactive oxygen species (ROS) increased TSPO levels on the surface of microglia and this effect was abrogated by a ROS scavenger. These results suggest that ROS production may alter the subcellular distribution of TSPO. Collectively, our findings suggest that in microglia, TSPO is associated with the major NOX2 subunits gp91phox and p22phox. We hypothesize that this interaction may regulate Cytb558 formation and modulate NOX2 levels, ROS production, and redox homeostasis in microglia.