Fig. 5.1
Role of peritoneal macrophages in inflammation
Another by-product of heme degradation by HO-1 is bilirubin, a powerful antioxidant. Bilirubin has also been found in higher levels in the macrophages of women with endometriosis [12]. Iron storage in macrophages also correlates with amounts of iron overload in the peritoneal fluid. Higher iron levels have been detected in the peritoneal fluid of patients with endometriosis; these iron levels have also been shown to correlate with the severity of the disease [14, 15].
Not only is free iron from heme degradation a source of increased iron levels in the peritoneal fluid of women with endometriosis, but macrophages are also able to release ferritin, which may add to the higher iron concentrations found in endometriosis patients [16, 17].
Transferrin, which ensures iron transport, also contributes to iron overload in the pelvic cavity. Transferrin can be incorporated into and expressed by ectopic endometrial cells in cases of iron overload. In endometriosis, peritoneal macrophages have more transferrin receptors than normal [18]. Studies have shown an association between increased levels of both ferritin and transferrin in women with endometriosis versus women without the disease, both in the peritoneal fluid and in peritoneal macrophages [11, 19]. High iron levels in cystic fluid and carcinomas have also been associated with endometriosis and possible carcinogenesis within the cysts due to the effects of iron and OS [20].
Lousse et al. reported that iron storage is significantly increased in peritoneal macrophages in women with endometriosis [11]. This increased iron storage correlated with iron overload in the peritoneal fluid. Van Langendonckt et al. found that iron deposits in ectopic endometrial lesions could be induced by the addition of red blood cells in menstrual effluent [21], showing that retrograde menstruation is one of the major contributors to iron accumulation in endometriosis. Defrère et al. reported that iron overload enhances the proliferation of ectopic endometrial cells in endometriosis [22]. While injection of erythrocytes and menstrual effluent resulted in the proliferation of endometrial lesions, it did not lead to the initial establishment of endometriosis. This is important because significant iron overload may only occur after endometrial lesions have already been established, but not during the initial stages of the disease when refluxed endometrial tissue is first attaching to areas in the pelvic cavity. These overall increases in iron accumulation may be due to higher degradation of red blood cells from retrograde menstruation, as well as a deficiency in iron metabolism in endometriosis patients.
In retrograde menstruation, there is an abundance of menstrual reflux into the peritoneal cavity. The refluxed erythrocytes are phagocytized by peritoneal macrophages, and as a result, they release Hb into the peritoneal cavity. Haptoglobin binds free Hb in the blood to prevent oxidative damage and the hemoglobin-haptoglobin complex is usually removed (Fig. 5.2). Free Hb is also digested by hemeoxygenase-1 (HO-1), and iron is subsequently released from the heme molecules. Ferritin is one of the metabolic byproducts of hemeoxygenase-1. It sequesters iron and prevents oxidative stress. Hemosiderin is a complex of ferritin that also stores iron within macrophages. Iron is usually transferred to ferritin and hemosiderin within macrophages to prevent iron-mediated damage, inflammation, and the production of excess amounts of ROS that may lead to oxidative stress (Fig. 5.2) [23].
Fig. 5.2
The role of iron in the pathophysiology of endometriosis. HO hemeoxygenase, CO carbon oxide
Iron accumulation can lead to numerous cytotoxic effects because it can disrupt the balance between free radical production and antioxidant defense, which leads to oxidative stress (OS) and, in turn, contributes to the pathogenesis of endometriosis. Therefore, iron-induced OS may trigger the chain of events resulting in the development and progression of the disease [24].
Iron overload caused by retrograde menstruation leads to accumulation of somatic mutations through Fenton reaction-mediated OS. The development of endometriosis is triggered by epigenetic disruption of gene expression along with environmental changes. There are three phases for development of endometriosis: genetic inheritance from parents, epigenetic modification in the female offspring, and iron overload, which is subjected to modulation later in life [1].
However, with an abundance of menstrual reflux with an overwhelmed peritoneal disposal system or along with a defective disposal system, iron levels exceed the sequestration capability of the macrophages, leading to iron overload. There is a resultant iron overload in the peritoneal environment, which in turn permits attachment and growth of the endometrial cells or fragments [25] and, ultimately, iron toxicity [23]. Iron overload may also occur when endometrial lesions bleed. This leads to the additional accumulation of erythrocytes in the peritoneal cavity [22] (Figs. 5.2 and 5.3). Iron levels in peritoneal macrophages of women with endometriosis tend to be higher than levels in macrophages from healthy women [11]. Studies have also shown that iron levels in the peritoneal fluid of women with endometriosis correlate with the severity of the disease [14, 15].
Fig. 5.3
Iron metabolism and binding in peritoneal cavity. Hb-Hp hemoglobin-haptoglobin, HO hemeoxygenase, CO carbon oxide
Van Langendonckt et al. showed that endometrial lesions can be induced in nude mice by injecting human menstrual tissue into the pelvic cavity, thereby mimicking menstrual reflux [21]. Defrère et al. found that while adding erythrocytes to the menstrual fluid injections did not lead to the establishment of endometrial lesions in mice, it did significantly increase the proliferation of the lesions [22]. These studies help confirm that menstrual reflux and iron metabolism within the peritoneal cavity play a role in the development of endometriosis and may be factors in oxidative damage associated with the presence of endometrial lesions. A disrupted peritoneum is not a requirement for endometrial tissue invasion [26].
5.4 Iron and Oxidative Stress
Iron-induced oxidative stress plays a fundamental role in the pathogenesis of endometriosis. Oxidative stress, secondary to influx of iron during retrograde menstruation, modifies lipids and proteins, leading to cell and DNA damage. Studies have demonstrated that hepatocyte nuclear factor (HNF-1β) overexpression in endometriotic foci increases the survival of endometriotic cells under iron-induced oxidative stress conditions possibly through the activation of forkhead box (FOX) transcription factors and/or endometriosis-specific expression of microRNAs. Endometriotic cells expressing HNF-1β also display cell cycle checkpoint pathways required to survive DNA damaging events [27].
The iron-induced ROS signals can contribute to carcinogenesis via estrogen-dependent pathways leading to endometrioid adenocarcinoma (EAC) or estrogen-independent clear cell carcinoma (CCC), thus supporting tumor progression and metastasis [28].
5.4.1 Fenton Reaction
Iron toxicity is mainly related to its ability to catalyze the production of a wide variety of damaging free radical species in the Fenton reaction, leading to deregulation of cellular processes, cell dysfunction, and eventually to apoptosis or necrosis through lipid peroxidation, protein, and DNA damage [29].
Iron acts as a catalyst in the Fenton reaction and generates wide range of free radicals such as hydroxyl radicals (OH) or the peroxynitrite anion (ONOO−), which is the product of reaction between NO and superoxide anion (O2−) (Fig. 5.4).
Fig. 5.4
Implications of Fenton reaction in endometriosis. ROS reactive oxygen species, NF-κβ nuclear factor kappa β, COX cyclooxygenase
5.5 Mechanistic Pathway of Iron Induced Oxidative Stress in Endometriosis
Endometriosis → Retrograde Menstruation → Iron → Oxidative stress.
5.6 Hemoglobin: Haptoglobin Complex, Hemopexin and Fenton Reaction
After phagocytosis of senescent erythrocytes, Hb may be catabolized by hemeoxygenase-1, or Hb may form a complex with haptoglobin. Haptoglobin is a protein that binds free Hb with a high affinity to prevent oxidative damage that can result from Hb overload [30]. The hemoglobin-haptoglobin complex can be cleared by parenchymal cells of the liver [31] although the complex may also be scavenged by peritoneal macrophages [32]. Binding of Hb by haptoglobin is a defense mechanism. Both Hb and haptoglobin can induce inflammation and oxidative stress [18, 19]. This defense mechanism still may be overloaded by Hb in the peritoneal cavity.
A Hb scavenger receptor, cluster of differentiation 163 (CD163) is expressed on monocytes and macrophages [33]. CD163 is a member of the cysteine-rich scavenger receptor family [34]. The CD163 scavenger receptor mediates endocytosis of Hb into peritoneal macrophages [34] and also mediates the degradation of the hemoglobin-haptoglobin complex [35]. The CD163-mediated uptake of iron through the hemoglobin-haptoglobin complex may explain iron accumulation within macrophages. The CD163 scavenger receptor present on macrophages also acts in coordination with haptoglobin [30, 36]. There are several scavenger receptors including CD163 and CD206 that are involved in both scavenging of hemoglobin with iron transfer into macrophages and the silent clearance of inflammatory molecules.
In endometriosis, peritoneal macrophages have been found to accumulate iron. Phagocytosis of red blood cells or endocytosis of the hemoglobin-haptoglobin complex leads to the origin of iron in macrophages. Heme, which is a product of catabolization of hemoglobin by HO, produces reactive iron. This free iron is then incorporated into macrophage ferritin or transferrin in the peritoneal fluid. Studies have reported that peritoneal macrophages in endometriosis patients exhibited high Tf receptor expression and were more likely to be saturated with Hp [18]. Endometrial lesions have also been found to synthesize and secrete Hp [37, 38]. Haptoglobin may act as an angiogenic or immunomodulatory factor [37]. This may lead to further vascularization of lesions and possible proliferation. Past studies have reported findings of increased haptoglobin in the peritoneal fluid of women with endometriosis [39]. However, other studies have reported no increase in Hp levels in endometriosis patients [40]. Haptoglobin may not be sufficient in binding Hb. Therefore, free Hb may be present in the peritoneal fluid and provide a source of heme that may become bound to hemopexin (HPX). Hemopexin is a glycoprotein that binds heme with the highest affinity of any protein. HPX scavenges heme that is released in the degradation of Hb by HO-1 and acts as another extracellular defense mechanism to protect the body from OS induced by free heme and iron molecules. Heme may cause damage through lipid peroxidation and the production of hydroxyl radicals [40]. The antioxidant capacity of HPX can also be overwhelmed by heme and iron overload in the peritoneal cavity [41].
HPX is also involved in cytoprotection and the prevention of iron-mediated damage, including the prevention of inflammation and proliferation of endometrial lesions [42]. Liver cells uptake heme and HPX in order to maintain levels of iron in the body along with Hb-Hp complexes. Hemopexin binds heme and transports it into the liver for degradation in the reticuloendothelial system. Blood serum levels of HPX have been used to indicate how much free heme is present in the blood. HPX levels in peritoneal fluid may also prove to be a marker of endometriosis. Macrophages are also able to uptake HPX-bound heme, but not in significant amounts except in cases of iron overload [23]. Studies have shown that hemopexin is significantly down-regulated in peritoneal fluid from patients with endometriosis [43].
5.7 Ferritin, Transferrin, and Hemosiderin
When heme-sequestering proteins experience iron overload, the degradation of Hb and heme by HO-1 releases free iron. Iron can be stored by the cells of endometrial lesions through the iron transporter protein transferrin within the peritoneal fluid, or the free iron can be stored in ferritin within macrophages [23]. Ferrous iron is able to catalyze the formation of ROS through the Fenton reaction, which produces hydroxyl radicals [44]. When free ferrous iron is released into the peritoneal fluid, it can be stored in ferritin, which oxidizes the ferrous iron to ferric iron and traps it within the shell of the protein [45].
Ferritin is an intracellular protein that stores and releases iron within macrophages. When ferritin does not have iron bound to it, it is called apoferritin. Because iron is redox-active, ferrous iron can be released by ROS from ferritin. Redox-active iron can lead to the generation of even higher amounts of ROS, continuing the cycle of iron release and cell damage by OS [1].
Hemosiderin is an iron-storing complex of ferritin, which also allows for iron accumulation within macrophages. The name hemosiderin can be used to describe clusters of iron deposits within cells, which may or may not include ferritin within the deposits [46]. Hemosiderin molecules may be found independently apart from ferritin in cases of iron overload [12]. Unlike ferritin, hemosiderin cannot be released from macrophages. However, hemosiderin-laden macrophages may be incorporated into endometrial lesions, resulting in the dark lesions seen in endometriosis [47].
Hephaestin is a membrane-bound protein that is essential for normal iron metabolism. It is a multi-copper ferroxidase protein that promotes the oxidation of ferrous iron to ferric iron [48]. Ferric iron can be bound to transport proteins such as transferrin and other plasma ligands [49]. When ferrous iron is oxidized by hephaestin, it can be taken up by transferrin and transported to the basolateral membrane [45]. This oxidization of iron allows the free iron to bind rapidly to transferrin and be transported to receptors on cells to prevent oxidative damage [49].
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