Iron Macrophages: Dance of Death and MMP Release in Intraplaque Hemorrhage

Atherosclerosis causes significant morbidity and mortality through vessel narrowing or occlusion, causing coronary heart disease and stroke.[1] While some atherosclerotic plaques are stable and lie dormant for many years, others can become unstable, leading to plaque rupture and thereby triggering thrombosis and vessel occlusion, the major cause of myocardial infarction and stroke.[1] [2] Identifying the factors that lead to plaque instability and finding therapies that target unstable atherosclerotic plaques is an important goal of cardiovascular medicine.

Intraplaque hemorrhage (IPH) is a cardinal feature of unstable atherosclerotic plaques and is believed to result from either the rupture of thin fibrous caps or the development of an abnormal, “leaky” vascular network within the atheroma, both of which allow the extravasation erythrocytes and the accumulation of their breakdown products, such as cholesterol, phospholipids, hemoglobin, hemin, and iron, within the plaque.[3]

In a recent study published in Thrombosis and Haemostasis, Li et al[4] describe a cascade of events following IPH that contributes to plaque instability. First, using a rabbit IPH model, the authors show a decrease in collagen staining and an increase in the expression of collagen-degrading matrix metalloproteases (MMPs) 2 and MMP9. Notably, the increase in MMP2 and MMP9 colocalized with plaque macrophages. Furthermore, the levels of hemin and iron where both increased following IPH and treatment of macrophages with purified hemin could upregulate MMP2 and MMP9 expression. These observations suggest that following IPH, hemin from damaged erythrocytes increases macrophage MMP2 and MMP9 expression, leading to collagen degradation and plaque instability. In considering how hemin might promote MM2 and MMP9 expression, the authors noted that intraplaque iron levels were increased following IPH and therefore investigated whether ferroptosis may contribute to the above-described phenomena ([Fig. 1]).

Fig. 1 Proposed mechanism of matrix metalloprotease (MMP) 2 and 9 release from plaque macrophages during intraplaque hemorrhage (IPH): Following an IPH, macrophages phagocytose red blood cells and their components. This results in increased iron (Fe³⁺), which when metabolized through the Fenton reaction, generates products that can induce peroxidation of membrane lipids causing cell death via ferroptosis. Lipid peroxidation and/or ferroptosis is suggested to phosphorylate and activate p38, through a yet to be described mechanism resulting in MMP2/9 release which can contribute to collagen breakdown, plaque rupture, thrombosis formation, and clinical consequences such as myocardial infarction and stroke.

Ferroptosis is a form of cell death triggered by iron-dependent lipid peroxidation (LPO).[5] [6] Ferroptosis has been implicated in several diseases, most notably in organ injury and neurodegeneration, but also in cardiovascular disease.[5] [6] Iron plays a key role in the initiation of LPO and increasing the pool of redox active iron can increase LPO and ferroptotic cell death.[5] [6] The primary means by which iron triggers LPO is through its role in the Fenton reaction, which generates highly reactive hydroxyl radicals (HO^•).[7] LPO is initiated when HO^• “attack” specific sites within polyunsaturated fatty acids (PUFAs), generating a carbon-centered lipid radical (L^•), which subsequently react with molecular oxygen to form a lipid peroxyl radical (LOO^•).[7] During the propagation phase of LPO, LOO^• remove H atoms from neighboring PUFAs within the membrane to form lipid hydroperoxides (LOOH).[7] This chain reaction of events can severely compromise membrane integrity, leading to ferroptotic cell death. To prevent excessive LPO and ferroptosis, cells possess the means to detoxify LOOH (via glutathione peroxidase [GPX]4) and lipid radicals (e.g., antioxidants such as α-tocopherol and ubiquinol).[5] [6]

First, Li et al show that macrophages treated with hemin in vitro causes LPO and cell death. These effects can be partially reversed by a ferroptosis inhibitor, ferrostatin-1, which works as a radical trapping antioxidant, and by iron chelation. Furthermore, ferrostatin-1 reversed hemin-induced increases in MMP2 and MMP9 expression. Collectively, these data support the hypothesis that increased intraplaque hemin and iron following IPH triggers LPO and ferroptosis in macrophages, and these events through p38 MAPK contribute to increased MMP2 and MMP9 expression. However, whether ferroptotic cell death is required to induce p38 activation and release MMP2 and MMP9 from plaque macrophages, or whether signals from LPO are sufficient to mediate MMP2/9 expression is unclear ([Fig. 1]). However, phosphorylated p38 was also reduced with the administration of ferrostatin-1, raising the question if LPO fragments can activate this pathway as reported in tumor infiltrating CD8⁺ T cells.[8] Nonetheless, to establish in vivo if LPO (ferroptosis) contributed to plaque instability following IPH, the authors administered ferrostatin-1 to Apoe deficient mice with spontaneous carotid artery IPH. Using this model, the authors first demonstrate the presence of fragmented erythrocytes as well as increased 4-hydroxynonenal (HNE) (HNE is a product of LPO) staining in macrophages within the plaque. Importantly, ferrostatin-1 decreased MMP2 and MMP9 expression, increased collagen expression, decreased plaque area, and increased the luminal area. Collectively, these results suggest that following IPH, increases in hemin and iron within intraplaque macrophages leads to LPO, and likely ferroptosis. These events lead to plaque progression and instability via an increase in collagen degrading MMP2 and MMP9.

These findings contribute to a growing body of literature that indicates a potential role for ferroptosis in the progression of atherosclerosis.[9] [10] [11] Of particular interest are recent studies showing that erythrophagocytosis promotes LPO and ferroptosis in plaque-resident macrophages, likely via an increase in intracellular iron levels, and thereby promotes atherosclerosis.[10] [11] Notably, these effects can be reversed by inhibitors of ferroptosis.[10] [11] Developing effective radical trapping antioxidants that work in vivo to limit excessive LPO and ferroptosis to prevent tissue damage is a major goal of the field. Whether such compounds may be effective in preventing plaque rupture, and thereby limiting the morbidity and mortality that occurs as a result of MI or stroke, will be an important future goal for the field.

Publication History

Received: 23 November 2023

Accepted: 30 November 2023

Article published online:
09 January 2024

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