During atherosclerosis, lipid-rich plaques are formed in large- and medium-sized arteries, which can reduce blood flow to tissues. This situation becomes particularly precarious when a plaque develops an unstable phenotype and becomes prone to rupture. Despite advances in identifying and treating vulnerable plaques, the mortality rate and disability caused by such lesions remains the number one health threat in developed countries. Vulnerable, unstable plaques are characterized by a large necrotic core, implying a prominent role for necrotic cell death in atherosclerosis and plaque destabilization. Necrosis can occur accidentally or can be induced by tightly regulated pathways. Over the past decades, different forms of regulated necrosis, including necroptosis, ferroptosis, pyroptosis, and secondary necrosis, have been identified, and these may play an important role during atherogenesis. In this review, we describe several forms of necrosis that may occur in atherosclerosis and how pharmacological modulation of these pathways can stabilize vulnerable plaques. Moreover, some challenges of targeting necrosis in atherosclerosis such as the presence of multiple death-inducing stimuli in plaques and extensive cross-talk between necrosis pathways are discussed. A better understanding of the role of (regulated) necrosis in atherosclerosis and the mechanisms contributing to plaque destabilization may open doors to novel pharmacological strategies and will enable clinicians to tackle the residual cardiovascular risk that remains in many atherosclerosis patients.

Gasdermin D (GSDMD) is the key executor of pyroptotic cell death. Recent studies suggest that GSDMD-mediated pyroptosis is involved in atherosclerotic plaque destabilization. We report that cleaved GSDMD is expressed in macrophage- and smooth muscle cell-rich areas of human plaques. To determine the effects of GSDMD deficiency on atherogenesis, ApoE^−/− Gsdmd^−/− (n = 16) and ApoE^−/−Gsdmd^+/+ (n = 18) mice were fed a western-type diet for 16 weeks. Plaque initiation and formation of stable proximal aortic plaques were not altered. However, plaques in the brachiocephalic artery (representing more advanced lesions compared to aortic plaques) of ApoE^−/− Gsdmd^−/− mice were significantly smaller (115 ± 18 vs. 186 ± 16 × 10³ µm², p = 0.006) and showed features of increased stability, such as decreased necrotic core area (19 ± 4 vs. 37 ± 7 × 10³ µm², p = 0.03) and increased αSMA/MAC3 ratio (1.6 ± 0.3 vs. 0.7 ± 0.1, p = 0.01), which was also observed in proximal aortic plaques. Interestingly, a significant increase in TUNEL positive cells was observed in brachiocephalic artery plaques from ApoE^−/− Gsdmd^−/− mice (141 ± 25 vs. 62 ± 8 cells/mm², p = 0.005), indicating a switch to apoptosis. This switch from pyroptosis to apoptosis was also observed in vitro in Gsdmd^−/− macrophages. In conclusion, targeting GSDMD appears to be a promising approach for limiting the transition to an inflammatory, vulnerable plaque phenotype.

Intraplaque (IP) angiogenesis is a key feature of advanced atherosclerotic plaques. Because IP vessels are fragile and leaky, erythrocytes are released and phagocytosed by macrophages (erythrophagocytosis), which leads to high intracellular iron content, lipid peroxidation and cell death. In vitro experiments showed that erythrophagocytosis by macrophages induced non-canonical ferroptosis, an emerging type of regulated necrosis that may contribute to plaque destabilization. Erythrophagocytosis-induced ferroptosis was accompanied by increased expression of heme-oxygenase 1 and ferritin, and could be blocked by co-treatment with third generation ferroptosis inhibitor UAMC-3203. Both heme-oxygenase 1 and ferritin were also expressed in erythrocyte-rich regions of carotid plaques from ApoE^−/−Fbn1^C1039G+/− mice, a model of advanced atherosclerosis with IP angiogenesis. The effect of UAMC-3203 (12.35 mg/kg/day) on atherosclerosis was evaluated in ApoE^−/−Fbn1^C1039G+/− mice fed a western-type diet (WD) for 12 weeks (n = 13 mice/group) or 20 weeks (n = 16–21 mice/group) to distinguish between plaques without and with established IP angiogenesis, respectively. A significant decrease in carotid plaque thickness was observed after 20 weeks WD (87 ± 19 μm vs. 166 ± 20 μm, p = 0.006), particularly in plaques with confirmed IP angiogenesis or hemorrhage (108 ± 35 μm vs. 322 ± 40 μm, p = 0.004). This effect was accompanied by decreased IP heme-oxygenase 1 and ferritin expression. UAMC-3203 did not affect carotid plaques after 12 weeks WD or plaques in the aorta, which typically do not develop IP angiogenesis. Altogether, erythrophagocytosis-induced ferroptosis during IP angiogenesis leads to larger atherosclerotic plaques, an effect that can be prevented by ferroptosis inhibitor UAMC-3203.

RIPK1 (receptor-interacting serine/threonine-protein kinase 1) enzymatic activity drives both apoptosis and necroptosis, a regulated form of necrosis. Because necroptosis is involved in necrotic core development in atherosclerotic plaques, we investigated the effects of a RIPK1^S25D/S25D mutation, which prevents activation of RIPK1 kinase, on atherogenesis in ApoE^−/− mice. After 16 weeks of western-type diet (WD), atherosclerotic plaques from ApoE^−/− RIPK1^S25D/S25D mice were significantly larger compared to ApoE^−/− RIPK1^+/+ mice (167 ± 34 vs. 78 ± 18 × 10³ µm², p = 0.01). Cell numbers (350 ± 34 vs. 154 ± 33 nuclei) and deposition of glycosaminoglycans (Alcian blue: 31 ± 6 vs. 14 ± 4%, p = 0.023) were increased in plaques from ApoE^−/− RIPK1^S25D/S25D mice while macrophage content (Mac3: 2.3 ± 0.4 vs. 9.8 ± 2.4%, p = 0.012) was decreased. Plaque apoptosis was not different between both groups. In contrast, pharmacological inhibition of RIPK1 kinase with GSK’547 (10 mg/kg BW/day) in ApoE^−/− Fbn1^C1039G+/− mice, a model of advanced atherosclerosis, did not alter plaque size after 20 weeks WD, but induced apoptosis (TUNEL: 136 ± 20 vs. 62 ± 9 cells/mm², p = 0.004). In conclusion, inhibition of RIPK1 kinase activity accelerated plaque progression in ApoE^−/− RIPK1^S25D/S25D mice and induced apoptosis in GSK’547-treated ApoE^−/− Fbn1^C1039G+/− mice. Thus, without directly comparing the genetic and pharmacological studies, it can be concluded that targeting RIPK1 kinase activity does not limit atherogenesis.