The aetiopathogenesis of ED is not clearly known. However, recently literature associated ED with tuberculosis, but still now the active organism was not cultivated. The disease still remains idiopathic, with no diagnostic test available in the lab. Direct evidence of infection is not made. Similar to homocysteine, Iron also act as an independent risk factor of cardiovascular diseases. Roest (23) observed increased iron stores leads to cardiovascular diseases similar to that of hyperhomocysteine. Sullivan (24) observed that increased iron stores leads to endothelial dysfunction and that elevated homocysteine is dependent on iron overload. This excess iron generates free hydroxyl radical and causes oxidative stress through Fenton reaction. Schiepers (25) observed that changes in both homocysteine and ferritin levels in cardiovascular disease. The serum ferritin is frequently used as a measure of iron stores. The present findings indicate iron storage since; there is an elevated level of ferritin and lowered transferrin in ED.
It is known that, red cell breakdown can occur outside or inside the vascular compartment in ED. Increased serum haemoglobin in ED indicates the vascular damage due to intra vascular and extra vascular hemolysis. Extra vascular hemolysis are occur due to phagocytosis whereas intravascular hemolysis as a result of mechanical injury and toxic factors. Heme is the prosthetic group of heme proteins such as haemoglobin, is an essential molecule plays a crucial role in cell differentiation and other functions. Free heme namely unbound heme can be toxic to cells, because it results in production of reactive oxygen species and causes cell damage. The absolute levels of Heme are regulated by its biosynthesis and catabolism. Heme biosynthesis is regulated by ALAS and its degradation to Fe, bilirubin and CO is catalyzed by HO, in this study, it is observed that both the enzymes are elevated in ED.
Heme oxygenase plays an important role in regulating the heme level by catalyzing the initial and rate limiting step of heme degradation and resulting in the formation of carbon monoxide, iron and bilirubin. Heme oxygenase exists as three isoforms; HO-1, HO-2 and HO-3. HO-1, the inducible 32-kDa isoform, HO-2, the constitutive 36-kDa isoform, and HO-3, has no activity and is not expressed in humans. The HO-1 is a member of the heat shock proteins, and its expression is influenced by hypoxia (26), heavy metals, ROS such as H2O2 (27), reactive nitrogen oxides (28), TNF α, interleukin β and interferon ϒ (29). The biological functions of HO-1 are associated against oxidative and cellular stress. HO-1 represents a crucial mediator of antioxidants and possesses anti-inflammatory and anti- apoptotic properties (30). L’Abbate et al., and Bharathselvi et al., (31, 32) have shown that induction of HO-1 was associated with a parallel increase in the serum levels of adiponectin, which has a well documented anti-inflammatory property. The peroxisome proliferator-activated receptor (PPAR-ϒ) regulates the expression of HO in human vascular cells (33).
Over expression of HO-1 contributes in the revascularization of damaged tissue. In terms of neovascularization, HO-1 having a pro-angiogenic, anti-inflammatory and anti-apoptotic enzyme in regulation of wound healing (34, 35). Product of HO activity the bilirubin is a powerful antioxidant thereby, protecting the retinal cells. However, during haemorrhage, the iron and bilirubin excessively produced and are neurotoxic, have deleterious consequences. HO-1 is an inducible enzyme whose activity increases in response to iron as well as heme, light, oxidative stress, and inflammation. The main condition for the initiation of neovascularization is hypoxia (36). Hypoxia-inducible factor activates several genes related to iron metabolism such as HO-1, endothelin-1, transferrin, transferrin receptor and ceruloplasmin (36).
HO, cleavage of the heme ring will release intracellular iron, which in turn increases the stimulation of ferritin. Dulak et.al observed that HO plays an important role in angiogenesis during hypoxia, similarly nitric oxide synthase, by VEGF production (37). Alternately VEGF can stimulate HO-1 to promote angiogenesis and inhibiting leukocyte adhesion and transmigration (17). In the present study, it’s observed that both HO and VEGF increased due to inflammation and non-inflammatory responses.
Hepcidin, a circulating peptide hormone is mainly synthesized by the liver hepatocytes, and also in eye plays a major role in regulating iron homeostasis in the body (38). The mature form is 25 amino acids with four inter subunit disulfide bonds. The massive iron overload found in hepcidin knock-out mice suggests that hepcidin is an iron stores regulator involved in communication of body iron status to the intestine and also in the retinal pigment epithelial cells (38). The mechanism of hepcidin activity depends on hepcidin interactions with ferroportin. Ferroportin is the only known mammalian cellular iron exporter. Hepcidin regulates post translational ferroportin expression (38). Hepcidin binds to ferroportin and causes its internalization and degradation in turn blocks the iron transport via ferroportin (38). Hepcidin is reported to be elevated in chronic inflammation condition, anemia, more specifically wherein iron accumulates inside the cells (39).
Hepcidin synthesized can be induced by inflammatory cytokine IL-6, inflammation, and infection (40)[Prentice, 2012 #102]. IL‐6 acts via its receptor and causes phosphorylation of signal transducer and activator of transcription 3 (STAT 3), STAT3 activation requires the presence of SMAD 4 to affect the HAMP gene expression (39). Under normal conditions HAMP gene expression is regulated by BMP/SMAD and STAT3 pathways. Another hepatocyte iron sensors activating hepcidin synthesis are hemochromatosis protein (HFE) and transferrin receptor 2 (TfR2) (39).
Due to an intracellular storage of iron, there is an increased production of hepcidin. In the present study, there is an increased storage of ferritin levels and up regulation of hepcidin. Inflammation leads to cellular iron sequestering through IL-6 up regulation of hepcidin. Iron can be export from the cell with the help of iron exporter ferroportin. In the present study, we also observed that hepcidin expression was increased and ferroportin was decreased in ED compared to controls. Interestingly cellular iron accumulation caused diminished ferroportin after hepcidin binding. There is an increased expression of hepcidin may be due to increased iron, inflammation and infection. Importantly, a cellular iron act as a cofactor of HIF, in the present study, it shows that there is an increased expression of HIF2α. A previous report says that hypoxia is a negative regulator of hepcidin expression (39), but in the present study, shows that HIF 2α expression was increased in ED may be due to inflammation and also by IL-6. The interaction between hepcidin and ferroportin binding is a key step to control an iron homeostasis.
From the present study, we conclude that increased serum and intracellular ferritin, heme and HO, hepcidin play an important role in the vasculitis. The present study establishes the role for iron in disease pathogen. In our study thus the increased levels of these proteins may be due to an infection and inflammation conditions. The real significance of these findings needs to be understood in an animal model or an in-vitro cell culture experiments.