Inhibition of GSDMD activation by Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and atherosclerotic lesion development in ApoE-/- mice

Pyroptosis is a pro-inflammatory form of cell death that can be triggered by the activation of gasdermin D (GSDMD) via inflammatory caspases such as caspase-1. Recent studies suggest that targeting GSDMD activation or pyroptosis may help reduce vascular inflammation and the progression of atherosclerotic lesions. The current study explored the therapeutic effects of inhibiting GSDMD activation using the novel GSDMD inhibitor N-Benzyloxycarbonyl-Leu-Leu-Ser-Asp(OMe)-fluoromethylketone (Z-LLSD-FMK), the specific caspase-1 inhibitor N-Benzyloxycarbonyl-Tyr-Val-Ala-Asp(OMe)-fluoromethylketone (Z-YVAD-FMK), and a combination of both, in ApoE-/- mice fed a Western diet starting at 5 weeks of age. The study also examined the efficacy of these inhibitors in bone marrow-derived macrophages (BMDMs).

In vivo, plaque formation, GSDMD activation, and caspase-1 activation were observed in the aortas of the mice, with progressive increases from 6 to 18 weeks of age, and marked elevations at 14 and 18 weeks. From 14 to 18 weeks, ApoE-/- mice were intraperitoneally administered Z-LLSD-FMK (200 µg/day), Z-YVAD-FMK (200 µg/day), a combination of both, or a vehicle control. Treatment significantly reduced lesion formation, macrophage infiltration, and the levels of vascular cell adhesion molecule-1, monocyte chemoattractant protein-1, and pyroptosis-related proteins, including activated caspase-1, activated GSDMD, cleaved interleukin (IL)-1β, and high mobility group box 1, in the aortas. No significant differences in plasma lipid levels were observed.

In vitro, treatment with these polypeptide inhibitors dramatically decreased the percentage of propidium iodide-positive BMDMs, as well as the release of lactate dehydrogenase and IL-1β, and the levels of pyroptosis-related proteins in both supernatants and cell lysates, which were elevated by lipopolysaccharide + nigericin. Notably, there were no significant differences between the Z-LLSD-FMK and Z-YVAD-FMK groups, and the combination of both inhibitors did not enhance the effects. These findings suggest that suppressing GSDMD activation with either Z-LLSD-FMK or Z-YVAD-FMK reduces vascular inflammation and atherosclerotic lesion development in ApoE-/- mice.