One of the mechanisms proposed for the deleterious effects of homocysteine is its ability to generate reactive oxygen species thereby producing oxidative stress (Figure 2). It is generally proposed that homocysteine, due to the presence of a thiol group, can rapidly auto-oxidize in circulation in the presence of ceruloplasmin, the major copper binding protein in plasma, to form homocystine and hydrogen peroxide (H₂O₂), thereby generating oxidative stress [15]. However, several recent reports indicate that transition metal catalyzed oxidation of homocysteine is not a facile process. In fact transition metal catalyzed oxidation of cysteine has been reported to be much faster than that of homocysteine [16] and although the concentration of cysteine is about 20–25 times higher than that of homocysteine it is usually not considered a risk factor for cardiovascular diseases [17]. Therefore, it seems unlikely that the deleterious effect of homocysteine is due to the generation of hydrogen peroxide via metal catalyzed auto-oxidation. However, homocysteine might indirectly result in oxidative stress by decreasing the transcription, translation [18] and catalytic activity of antioxidant enzymes like glutathione peroxidase (GPx) and superoxide dismutase (SOD) [19]. Homocysteine treated bovine aortic endothelial cells showed a significant decrease in glutathione peroxidase activity. The effect of Homocysteine on enzyme activity is demonstrated by compensatory effect of GPx-1 over expression on the adverse effects of homocysteine on endothelial function [20]. Nonaka et al [21] reported that homocysteine decreases the secretion and expression of extra cellular superoxide dismutase (EC-SOD), the most abundant isozyme of SOD, in the vascular wall in rat vascular smooth muscle cells.

Hyperhomocysteinemia has also been reported to be associated albeit indirectly with hypoxic conditions. Supporting this is the expression of Cap43 [that codes for a 43 kDa protein associated with hypoxia in endothelial cells (EC)] in cells treated with homocysteine. Hypoxia in alveoli leads to damage of capillary wall, a condition predisposing for atherosclerosis. Furthermore, it has also been shown that there is a decrease in the MAT1A transcription and mRNA stability in cultured hepatocytes exposed to hypoxic conditions [22].

Elevated levels of homocysteine have been reported to decrease the bioavailability of endothelial nitric oxide. Under normal condition Nitric oxide (NO) exerts anti-atherosclerotic effect through various mechanisms (Table 3). The NO produced by endothelium is known to decrease in response to elevated levels of homocysteine [23]. Decrease in the bioavailability of nitric oxide due to increased homocysteine concentration is perceived to cause vasoconstriction thus leading to cardiovascular disorders. Interestingly, the bioavailability of NO is decreased in hyperhomocysteinemic condition despite normal expression of eNOS [24]. The decrease in the bioavailability of nitric oxide in hyperhomocysteinemic conditions may be attributed to the formation of S- nitrosohomocysteine formed under physiological conditions (Figure 2). NO can also rapidly react with molecular oxygen and other oxygen free radicals to form peroxynitrites (ONOO^-) [25-27]. Furthermore, endothelial tetrahydrobiopterin, a critical co-factor for the endothelial nitric-oxide synthase (eNOS) is also a target for oxidation by ONOO^- and its oxidation results in formation of trihydrobiopterin radical (BH3^•) and consequently decrease NO production [28,29]. Thus, elevated levels of homocysteine may lead to the accumulation of reactive oxygen species due to the decreased activity of antioxidant enzymes and these oxygen radicals could then potentially inactivate NO resulting in vasoconstriction.

Another potential mechanism for the decreased bioavailability of NO in hyperhomocysteinemic states is the increased generation of asymmetric dimethylarginine (ADMA), an analogue of L-arginine, which is a competitive inhibitor of eNOS [30]. ADMA also promotes the "uncoupling" of eNOS (Figure 2) leading to increased production of superoxide & other reactive oxygen species, which may cause further decrease in availability of NO. ADMA is produced during degradation of proteins containing methylated arginine residues by protein arginine N- methyltransferases (PRMTs) [31]. The increased SAM dependent generation of these methylated proteins, results in both increased production of ADMA and increased generation of homocysteine [32]. The inhibition of endothelial nitric oxide synthesis by ADMA impairs cerebral blood flow, which may contribute to the development of Alzheimer's disease [33].

Furthermore, one of the mechanisms proposed for the anti-thrombotic effect of NO is its ability to inhibit the expression of the prothrombotic protein PAI-1. It has also been shown that NO released from activated platelets inhibits the recruitment of platelets to the growing thrombus [34]. Thus decrease in NO concentration may result in increased expression of PAI-1 and platelet aggregation leading to thrombosis.

Intracellular oxidative stress may be either due to excessive generation of reactive oxygen species or to decreased ability of cells to scavenge the reactive oxygen species leading to its accumulation. We propose that homocysteine-induced oxidative stress is primarily due to the decreased ability of the cells to detoxify H₂O₂ & other lipid peroxides due to decreased activity of intracellular antioxidant enzymes. Furthermore, decreased bioavailability of nitric oxide may lead to the increased expression of pro-inflammatory cytokines and PAI which can potentially lead to cardiovascular diseases.

The major process linking levels of homocysteine with apoptosis and inflammatory pathway is the Endoplasmic Reticulum (ER) stress (Figure 3, branch 3,) and c-myc mediated signaling (Figure 3, branch 2). Endoplasmic Reticulum is the destination for secretary and extracellular proteins. It also serves as a site of calcium storage, calcium signaling, and biosynthesis of steroids, cholesterol & other lipids. The ER has high level of numerous resident chaperone proteins such as glucose-regulated proteins, GRP-78 & GRP-94, which under normal conditions are required for proper protein folding prior to export, to their destination. However, during energy deprivation these proteins initiate signal of ER stress, a condition in which unfolded & misfolded proteins accumulate [35,36]. Homocysteine has been shown to alter the cellular redox state resulting in ER stress [37]. Homocysteine increases the expression and synthesis of GRP78, a glucose-regulated protein that is induced during ER stress. Cells respond to ER Stress by various processes, prominent among these is a process known as unfolded protein response (UPR) mediated by ER-resistant trans-membrane protein kinase (IRE1) [38]. Furthermore, Homocysteine was found to induce the expression of glutamic acid decarboxylase (GADD45, GADD 153), ATF4 (Figure 3, branch 3) and YY1 [[38], Figure 3, branch 4] as well as RTP and HERP [39,40]. Interestingly, in a separate study it has been reported that inducers of GRP78 also increase the expression of these genes [41]. The GADDs also link homocysteine levels with ER stress and alterations in cell growth and proliferation. In addition, YY1, a member of GL1 zinc finger family enhances the transcription activation of GRP78 promoter under a variety of ER stress conditions [42,43]. Increased expression of YY1 mediates the stress signal from ER to nucleus.

However, exposure to excess ER stress results in apoptotic cell death. ER stress activates c-Jun N-terminal kinases (JNKs) that regulate gene expression via phosphorylation and activation of transcription factors such as c-JUN. The activation of JNK is mediated by TNF receptor-associated factor-2 (TRAF2), which transduce signals from IREs that act as stress sensors and initiates UPR [44]. TRAF2 activates the apoptosis-signaling kinase (ASK1) or MAPKKK (mitogen activated protein kinase kinase kinase). Activation of MAPKKK leads to activation of JNK protein kinase that in turn causes apoptosis [45]. The TRAF1 binds to the TRADD (TNFR-Associated Death Domain), which recruits the activated caspase 8 initiating a proteolytic cascade subsequently resulting in apoptosis. Furthermore, caspase 8 also leads to release of pro-apoptotic factor cytochrome C [46]. Homocysteine may induce oxidative stress and apoptosis through an NADPH oxidase and/or JNK-dependent mechanism(s) [47]. Extra cellular adenosine (Ado) along with homocysteine (Ado/Hcy) causes apoptosis of cultured pulmonary artery endothelial cells through the enhanced formation of intracellular S-adenosylhomocysteine (Figure 3, branch 1). SAH inhibits isoprenylcysteine carboxylmethyltransferase (ICMT), which results in decrease of Ras methylation and activation of downstream signaling molecules resulting in apoptosis. ICMT catalyzes the posttranslational methylation of isoprenylated C-terminal cysteine residues found in many signaling proteins such as small monomeric G proteins [48,49]. Similarly high concentration of adenosine results in apoptosis of L1210 lymphocytic leukemia cells. Apoptosis in these cells was preceded by an early but transient expression of the proto-oncogene c-myc [50].

Expression of c-myc sensitizes cells to a wide range of pro-apoptotic insults that include DNA damage, hypoxia and nutrient deprivation (Figure 3, branch 2). The pro -apoptotic effect of c-myc is mediated through the release of cytochrome C into the cytosol [51]. Holocytochrome C interacts with apoptotic protease activating factor (APAF-1), which then recruits and activates procaspase 9. This ternary complex triggers the autocatalytic processing of caspase 9 and subsequently activates caspase 3. However inhibition of CD95 and P53 signaling pathway does not block this release, but activation of the caspase dependent apoptotic machinery requires cooperation between c-myc induced cytochrome C release and CD95 signaling. Thus, c-myc induction leads to release of cytochrome C to the cytosol recruiting the cells to other apoptotic triggers like CD95 pathway or p53 activation. C-myc might also induce apoptosis through active response factor (ARF) expression via activation of P53 [52]. Recently it has been shown that homocysteine induced apoptosis in human umbilical vein endothelial cells is correlated with p53 dependent Noxa expression [53]. The expression of the Noxa gene involves direct activation of its promoter by p53. Interestingly, the activity of p53 is regulated through lysine methylation. Methylated p53 is restricted to the nucleus and has increased stability. The "hyper-stabilization" and activation of p53 result in cell cycle arrest and apoptosis [54]. The methyltransferase activity is critical for p53 dependent apoptosis. Thus, it can be perceived that in hyperhomocysteinemic state p53 lysine methylation could be inhibited. Homocysteine could also potentially inhibit endothelial cell growth by inhibiting the expression of cyclin A mRNA. Apart from cyclin A associated kinase activity, cyclin dependent kinase (CDK2) activity was also significantly inhibited [55]. Interestingly, stress induced activation of P53 promotes transcription of P21, which in turn binds to CDKs and leads to blocking of the G1 to S phase transition during cell cycle.

Homocysteine affects mitogenesis in a cell type specific manner. Although elevated levels of homocysteine lead to apoptosis and has growth inhibitory effect on endothelial cells, it leads to proliferation of smooth muscle cells eg. homocysteine enhances AP-1 activity in A7r5 aortic smooth muscle cells thus influencing cell proliferation [56]. In a recent report it was shown that elevated levels of homocysteine result in increased AP-1 nuclear protein binding, cell DNA synthesis and proliferation in mesangial cells by increasing Erk activity via a calcium-dependent mechanism [[57], Figure 3, branch 5]. Furthermore, homocysteine has been reported to up regulate the expression of VEGF mRNA in pigmented human endothelial cell line via ATF4 mediated activation. [58]. The cell survival signal from VEGF is mainly brought about by P13-mediated activation of Akt/PKB. The downstream targets for Akt/PKB pathway inhibit apoptosis. Furthermore, VEGF also leads to the induction of Raf-MEK -ERK pathway in human umbilical endothelial cells (HUVECs) relating to cell survival [59].

Apart from activating the unfolded protein response, homocysteine-induced ER stress also activates the sterol regulatory binding proteins (SREBPs). Homocysteine induces the expression of sterol regulatory element binding protein-1 (SREBP1, Figure 3), an ER membrane bound transcription factor, in cultured vascular endothelial cells and human hepatocyte leading to increased biosynthesis and uptake of cholesterol, triglycerides and accumulation of intracellular cholesterol [60,61]. Normally the expression and activity of SREBPs is regulated by SREBP cleavage activation protein (SCAP). However, it is believed that homocysteine circumvents this mechanism, maintaining the cells in sterol-starved state although lipids continue to accumulate.

Thus by mapping the genes (identified using literature based search) in appropriate pathway, we show that elevated levels of homocysteine cause the up regulation of ER stress proteins resulting in apoptosis. Homocysteine might also mediate apoptosis via P53 mediated pathway or by inhibition of methyl transferases like ICMT. Furthermore, ER stress also leads to altered lipid metabolism which may lead to cardiovascular disorders. Thus, homocysteine-induced ER stress emerges as the common pathway that relates to apoptosis and atherosclerosis. In this context it needs to be mentioned that homocysteine can potentially cleave critical protein disulfide bonds resulting in the alteration of structure and/or function of the protein [62-64]. It can be perceived that this might also lead to protein misfolding /unfolding which is a hallmark of ER stress.

During vascular injury, tissue factor, an integral membrane glycoprotein that is tightly associated with phospholipids, form a complex (1:1) with factor VII thereby initiating the coagulation cascade (Figure 4). Homocysteine can enhance the pro-coagulant activity in a number of ways. Elevated homocysteine levels have been reported to increase the cellular tissue factor activity [65]. Mann et al [63] also suggested that homocysteine rapidly incorporates into factor V resulting in impaired inactivation of factor Va by activated protein C (APC). Binding of homocysteine to factor V however did not have any effect on the conversion of factor V to factor Va. APC, a vitamin K dependent protein is formed by the action of thrombin on protein C in the presence of a membrane bound cofactor, thrombomodulin. Interestingly, homocysteine has also been shown to inhibit the cofactor activity of thrombomodulin [66]. Thus homocysteine impairs the thrombomodulin-APC anticoagulant pathway by inhibiting the cofactor activity of thrombomdulin resulting in the decreased formation of APC and also by inhibiting the inactivation of factor Va by APC. Furthermore, homocysteine also affect another important endothelial anticoagulant pathway viz. endothelial cell heparin-like glycosaminoglycans-antithrombin III anticoagulant mechanism [67]. It has been reported that incubation of endothelial cells with homocysteine suppresses the amount of antithrombin III binding to cell surface. Homocysteine also reduces the cellular binding for tissue plasminogen activator and enhances the plasminogen activator inhibitor-1 (PAI-1) gene expression and secretion from vascular endothelial and smooth muscle cells by a mechanism independent from paracrine-autocrine activity of TGF beta and TNF alpha. Hajjar et al also suggested that endothelial cells treated with homocysteine resulted in selective reduction in cellular binding sites for t-PA. This 65% decrease in binding was associated with a 60% decrease in cell-associated t-PA activity [64]. Thus homocysteine may promote prothrombotic state.

Thus, it can be perceived that elevated homocysteine levels will lead to prothrombotic state by enhancing the pro-coagulant pathway and/or suppressing the anticoagulant pathways.

Atherosclerosis is a chronic inflammatory disease of the artery, in which deposits of fatty substances, cholesterol, cellular waste products, calcium and other substances build up in endothelial layer of artery [68]. Apart from the conventional risk factors for atherosclerosis, elevated level of homocysteine is now considered to be an independent risk factor for cardiovascular diseases. Several mechanisms have been proposed for the homocysteine induced cardiovascular disease including altered lipid metabolism, cholesterol dysregulation, modulation of extracellular matrix protein expression, inflammatory response and oxidative stress. From the genes obtained after literature mining we found that homocysteine might induce atherosclerosis via one or more of the pathways depicted in Figure 5. The various interlinked pathways that contribute to the complex phenotype of atherosclerosis are outlined below.

Elevated levels of homocysteine have been associated with Alzheimer disease (Figure 7). A characteristic feature of the disease is the accumulation of amyloid beta (Aβ) peptide and formation of Amyloid plaques and neurofibrillary tangles in the brain. Although the mechanism of neurodegeneration has not yet been completely elucidated, it is reported that increased calcium levels in the cytosol, increased generation of reactive oxygen species, hyper phosphorylation of tau proteins and apoptosis are the important hallmarks of this disease [109,110]. Increased surface phosphatidyl serine (PS) is an early marker of neuronal apoptosis [111]. The involvement of reactive oxygen species is supported by the fact that high concentrations of copper have been found in the vicinity of Aβ amyloid deposits [112]. Moreover, high levels of antioxidant enzyme such as heme oxygenase-1, SOD-1, catalase and GPX were observed in plaque of brain tissue. Homocysteine also increases the levels of calcium and is known to generate reactive oxygen species especially in the presence of transition metal ions and thus it is not surprising that AD patients show elevated levels of homocysteine. Seshadri et al. found elevated levels of homocysteine prior to disease onset and suggested that the risk of AD increases by about 40% for every 5 μM increase in the concentration of homocysteine [113].

AD patients have elevated levels of homocysteine and decreased levels of SAM. This is believed to alter the DNA methylation status and hence gene expression in AD patients. This hypothesis is supported by the observation that SAM when added to human neuroblastoma SK-N-SH cells in culture, down-regulates expression of PS I gene coding for presenilin, a key factor for Aβ formation in AD due to methylation of its promoter [114]. Similarly, there are other studies suggesting that levels of SAM, folate and vitamin B12 influence DNA methylation of the genes that are involved in the Aβ formation [115]. Furthermore, it has also been reported that Herp, a homocysteine responsive protein, up regulated during ER stress, regulates PS-mediated amyloid beta generation presumably by binding to PS [116].

Homocysteine acts as an agonist and a partial antagonist at the glutamate binding site of the NMDA and the glycine-binding site of the receptor respectively. Under physiological conditions, when the concentration of glycine is normal, the neurotoxicity of homocysteine is observed at a very high concentration (millimolar range). However, under pathological conditions, such as in stroke or trauma where glycine levels in the brain is elevated, neurotoxicity of homocysteine is observed even at very low concentrations of homocysteine (10–100 μM) as the neurotoxic attributes of homocysteine exceeds its protective activity [117]. Under physiological conditions excitation of glutamate receptors initiates the stimulation of lipases and phospholipases with the generation of second messengers that are necessary for normal cell function. However, over stimulation of glutamate receptors leads to excessive calcium entry, abnormal phosphorylation and proteolysis. Thus, increase in the concentration of homocysteine probably results in the over stimulation of glutamate receptors resulting in increased calcium influx. The neuronal damage is due to excess calcium influx and also the accumulation of reactive oxygen species.

Thus, we propose that homocysteine either by inducing oxidative stress or ER stress might lead to apoptosis which in turn may result in neurological disorders. Alternatively homocysteine might act on glutamate receptors triggering a cascade of events that might result in the disease.