K. R. Rebello1,2, Y. Li1,2, A. Chakraborty1,2, L. Zhang1,2, C. Zhang1,2, Y. Li1,2, Y. H. Shen1,2, S. A. LeMaire1,2 1Baylor College Of Medicine, Division Of Cardiothoracic Surgery, Houston, TX, USA 2Texas Heart Institute, Houston, TX, USA
Introduction:
Although smooth muscle cell (SMC) loss is a key feature of aortic degeneration during formation of aortic aneurysms and dissections (AAD), the mechanisms driving SMC death are unclear. Pyroptosis involves pore formation in the cell membrane after gasdermin D (GSDMD) cleavage via caspase-1 (CASP1). We have shown that pyroptosis is active in AAD formation. Prior studies indicate that cytosolic DNA and its sensor cGAS (cyclic GMP-AMP synthase)-STING (stimulator of interferon genes) signaling are critical in SMC destruction. While mitochondrial damage in AAD releases mitochondrial DNA (mtDNA) into the cytosol, it is unknown whether mtDNA triggers pyroptosis. We examined the hypothesis that cytosolic mtDNA initiates the cGAS-STING pathway, which promotes GSDMD-induced pyroptosis. We also hypothesized that GSDMD can create pores in the mitochondrial membrane, generating a perpetuating cycle that promotes mitochondrial damage and cell death.
Methods:
In primary human vascular SMCs, we examined the direct effect of mtDNA release using rotenone and menadione, known mitochondrial damage inducers, to trigger the cGAS-STING pathway and activate GSDMD. Co-immunoprecipitation (co-IP) and immunofluorescence (IF) studies were used to evaluate interactions between STING, GSDMD, and CASP1. IF assays were done to show the presence of cytosolic mtDNA after treatment with both mitochondrial damage inducers. Cells underwent oxidative stress with 500μM H2O2 to induce GSDMD cleavage and pyroptosis in IF assays, and co-stained with TOM20, a mitochondrial marker.
Results:
We observed that treating human vascular SMCs with rotenone and menadione induced the cleavage and activation of GSDMD (cleaved N-terminal band present) that was associated with cytosolic mtDNA leak and activation of the cGAS-STING pathway (phospho-STING band present). Co-IP assays revealed that STING interacted with GSDMD and CASP1 (Fig 1A). Interestingly, IF studies showed that stressing SMCs with 500μM H2O2 induced translocation of the active N-terminal of GSDMD not only to the cell membrane, but also to the nucleus and mitochondria (indicated by its co-localization with mitochondrial membrane marker TOM20, Fig 1B).
Conclusion:
Our findings suggest that GSDMD-mediated pyroptosis can be triggered by mtDNA release secondary to mitochondrial damage via the cGAS-STING pathway. We show that oxidative stress induces active GSDMD to travel to the cell membrane, mitochondrial membrane, and nucleus. Pyroptosis may be potentiated by the cGAS-STING pathway, as STING can bind GSDMD and CASP1, to promote GSDMD cleavage. Future studies are needed to evaluate the effect of GSDMD in murine aortic disease models and to ascertain the nature of the STING/pyroptosis interaction.
