Programmed cell death in atherosclerosis and vascular calcification

Abstract

The concept of cell death has been expanded beyond apoptosis and necrosis to additional forms, including necroptosis, pyroptosis, autophagy, and ferroptosis. These cell death modalities play a critical role in all aspects of life, which are noteworthy for their diverse roles in diseases. Atherosclerosis (AS) and vascular calcification (VC) are major causes for the high morbidity and mortality of cardiovascular disease. Despite considerable advances in understanding the signaling pathways associated with AS and VC, the exact molecular basis remains obscure. In the article, we review the molecular mechanisms that mediate cell death and its implications for AS and VC. A better understanding of the mechanisms underlying cell death in AS and VC may drive the development of promising therapeutic strategies.

Fig. 1. Apoptotic signaling in VC.

Apoptosis can be triggered by intrinsic and extrinsic pathways. BH3-only proteins have crucial roles in intrinsic apoptosis, which can bind to anti-apoptotic proteins and inhibit their function, thereby inducing mitochondrial outer membrane permeabilization (MOMP). Afterward, released cytochrome c binds apoptotic peptidase activating factor 1 (APAF1) and leads to the formation of apoptosome. The caspase 9 is activated in this complex, in turn, it activates the executioner caspase 3/7. The extrinsic apoptotic pathway is initiated by ligand‐receptor binding, and then these death receptors can recruit caspase-binding adapter proteins to form the “death-inducing signaling complex (DISC)”, which activates caspase 8 and downstream effector caspases 3 and 7. The executioner caspases further cause internucleosomal DNA fragmentation, membrane blebbing, and apoptotic body formation. Macrophages can recognize the ‘eat me’ signals on the surface of these bodies, such as phosphatidylserine (PS), to mediate efferocytosis. Whereas, lipid metabolism disorder and calcium-phosphate imbalance can give rise to phagocytic dysfunction of macrophages and increased release of pro-calcific matrix vesicles. Furthermore, apoptotic bodies serve as nucleation points for calcium crystals with the deposition of hydroxyapatite. Together, these changes lead to vascular mineralization.

Fig. 2. Necroptotic signaling in AS.

Necroptosis is commonly induced by the binding of tumor necrosis factor (TNF) to its TNF receptor 1 (TNFR1). Afterward, TNFR1 recruits the adaptor protein TRADD which functions as a platform for recruiting other proteins and forming complex I. The deubiquitination of receptor-interacting protein kinases 1(RIPK1) results in the formation of complex IIb, activating the necroptotic signaling cascade. Here, the autophosphorylation of RIPK1 facilitates the phosphorylation and activation of RIPK3, which in turn mediates the phosphorylation and conformational changes of mixed-line kinase domain like (MLKL), eventually causing plasma membrane rupture. In early atherosclerotic diseases, high levels of RIPK1 drive NF-κB-dependent inflammation and the activation of ECs. Activated ECs secrete more adhesion molecules to attract and recruit monocytes and other inflammatory cells. Next, ox-LDL induces RIPK3 expression in macrophages and foam cells, promoting cellular necroptosis and the forming of necrotic lipid pool. Ultimately, this leads to plaque instability and rupture, as well as a variety of cardiovascular events.

Fig. 3. Pyroptotic signaling in AS and VC.

Cell recognizes a variety of stressors, such as DAMPs, PAMPs, uric acid crystals, toxins, phosphate, reactive oxygen species (ROS), etc., to undergo inflammasome formation and pyroptosis. Here, inflammasome formation requires at least two steps including priming and activation. Canonical NLRP3 gene transcription is triggered by the pattern recognition receptors (for example, TNFR, TLR, IL-1R) located at the cell surface. Then, the inhibitor of NF-κB (IκB) is phosphorylated and activated, and the functional NF-κB is released into the nucleus. As a result, the expression of pro-IL, pro-caspase11, and the inflammasome components (NLRP3, pro-caspase1, and ASC) is upregulated. The inflammasome is assembled. However, its activation mechanism remains largely unknown. Next, activated caspase-1 cleaves the cytokine precursors pro-IL-1β and pro-IL-18 into mature proinflammatory factors IL-1 and IL-18. In addition, it also mediates the cleavage of GSDMD to form the N-terminal pore-forming domain, which translocates to the cellular membrane, where it forms membrane pores and drives the release of pro-inflammatory cytokines. Persistent stimulation of the inflammatory leads to ECs damage, increased ROS generation, enhanced adhesion and migration of monocytes, foam cell necrosis, and VSMC proliferation and osteogenic differentiation, which facilitates AS and VC.

Fig. 4. Autophagic signaling in VC.

Autophagy is activated by multiple stressors. Subsequently, the uncoordinated-51-like protein kinase 1/2 (ULK1/2) complex dissociates from the mammalian target of rapamycin complex 1 (mTORC1) and becomes activated to initiate the formation of a phagophore. Phosphatidylinositol-3-phosphate (PI3P) promotes autophagosomal membrane nucleation. After, the elongation of the phagophore involves two ubiquitin-like conjugation systems: the Atg5-Atg12-Atg16L and the microtubule-associated protein 1 light chain 3-phosphatidylethanolamine (LC3-PE). This leads to the formation of autophagosomes. Finally, autophagosomes fuse with lysosomes to form autolysosomes where cargoes are degraded by lysosomal enzymes. Autophagy deficiency or lysosomal dysfunction promotes VC. The calcification precursors like calcium and phosphate are formed or processed in the endosome, autophagosomes, or autolysosomes, which participates in the regulation of intracellular calcium and phosphate homeostasis. Hydroxyapatites enter the cell by endocytosis, via endosomes. Then these endosomes fuse with autophagosomes and are transported to autolysosomes where they can be degraded or released into the ECM by packaging in the MVs. Impairment of lysosome function results in increased secretion of pro-calcific extracellular vesicles, promoting VC.

Fig. 5. Ferroptotic signaling in AS and VC.

Iron-dependent phospholipid peroxidation and reduced antioxidant capacity lead to cell death. Fe²⁺ provided by the labile iron pool (LIP) reacts with hydrogen peroxide (H₂O₂) to form the hydroxyl radical (OH·) through Fentons reaction. A carbon-centered lipid radical (PL·) is generated as PUFAs donates hydrogen to (OH·). Then, (PL·) reacts with molecular oxygen (O₂) to yield a phospholipid peroxyl radical (PLOO·) that abstracts hydrogen from adjacent PUFAs to generate a phospholipid hydroperoxide (PLOOH) and a new (PL·), initiating another lipid radical chain reaction. In enzymatic lipid peroxidation, the generation of PLOOH results from the dioxygenation of PUFAs by lipoxygenases (LOX). PLOOH in the presence of Fe²⁺ can be converted to the alkoxyl phospholipid radical (PLO·), triggering the propagation of lipid peroxidation. In addition, PLOOH can decompose to reactive toxic aldehydes such as 4-hydroxy-2-nonenals (4-HNEs) or malondialdehydes (MDAs) that induce cellular damage. Reduced antioxidant capacity is another important cause of ferroptosis. The inhibition of system Xc⁻ results in the GSH depletion and resultant inactivation of GPX4. Increased lipid peroxidation causes mitochondrial dysfunction, which in turn enhances ROS production and aggravates peroxidative damage. Also, PUFAs mediate inflammatory cascades. These adverse environmental factors together contribute to the progression of AS and the occurrence of VC.