Cardiovascular system consists of three components: heart, blood vessels and blood, and participates in maintaining of internal body homeostasis in many aspects: transport of oxygen, carbon dioxide, nutrients, metabolism waste products, and hormones to and from every single cell in the body. The blood vessels are composed of three separated layers: intima, media and adventitia. The intima consists of single layer of endothelial cells that have crucial role in regulation of blood flow. Media is dominantly composed of vascular smooth muscle cells, and represents the thickest layer. The adventitia is outer layer made of connective and fat tissue that have protective role of other inner layers. Blood vessels differ depending on part of circulation to which it belongs (arterial, capillary or venous), or of specific needs and functions of tissues and organs in which it is located.
Blood vessels are actively involved in the regulation of blood pressure and blood flow by summarizing the effects of the autonomic nervous system, various hormones and endothelial derived factors on changes of diameter. The vascular dilatation caused by shear stress of the blood is mediated by production of endothelium-derived relaxing factor – nitric oxide (NO) [1]. NO is synthesized by NO synthase and diffuses through cell membranes to the vascular smooth muscle cells, increases production of cyclic GMP and induces the relaxation.
Hyperhomocysteinemia (HHcy) is used as a predictive risk factor for cardiovascular disorders, the stroke progression, screening for inborn errors of Met metabolism, and as a supplementary test for vitamin B12 deficiency [2]. Hyperhomocysteinemia is associated with production of different ROS: superoxide anion (O2-), hydroxyl radical (•OH), peroxynitrite (ONOO-), hydrogen peroxide (H2O2), as well as other peroxides and hypochlorous acid, and their organic analogues [3]. Within the investigation where the coronary and mesenteric arteries were incubated with methionine it has been revealed the role of angiotensin- converting enzyme (ACE) and angiotensin II (ANGII) signaling pathway in activation of NADPH oxidase and increase in O2- production [4]. The link between ACE, ANGII signaling and NADPH oxidase have been noticed earlier, but in this study it was described the role of homocysteine in activation of ACE [5]. Namely, homocysteine induces homocysteinylation of ACE, which in turn has greater activity of this enzyme, followed by increased transduction by ACE/ANG II/AT1R signaling pathway, and of and of O2-.
On the other hand, homocysteine induces increased expression of different forms of NADPH oxidase. NOX4 is an isoform of NADPH oxidase highly represented in the kidney, and incubation of tubular cells with homocysteine showed increased production of O2- [6]. Proposed underlying mechanism was incrementing the expression of NOX4 by homocysteine. Increased production of O2- leads to decreased bioavailability of nitric oxide (NO), due to reaction of these two molecules and production of highly reactive peroxynitrite (ONOO-). NO is synthesized by three isoforms of enzyme nitric oxide synthase (NOS): endothelial NOS (eNOS or NOSI), inducible NOS (iNOS or NOSII) and neuronal NOS (nNOS or NOS III). eNOS and nNOS are constitutive enzymes and their activity is regulated by changes in Ca2+ content in the cytoplasm. All three isoforms generate NO from L-arginine in the presence of O2 and NADPH. Also as cofactors are necessary flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and tetrahydrobiopterin (BH4). BH4 is crucial for NOS function because it binds NOS monomers to form dimers which contain two reductase domains ‘coupled’ to another pair of oxygen domains. NOS monomers generate O2- instead of NO, and this ‘uncoupled’ enzyme represents a ROS producer. The classical signaling pathway of NO includes activation of soluble guanylyl cyclase and production of cyclic guanosine 3’,5’-monophosphate (cGMP). NO acts in autocrine or paracrine manner. In study on HUVECs increased homocysteine induced decrement in NO production, and also an increase in production of endothelin-1 (ET-1), as one of the most potent vasoconstrictors. On the other hand homocysteine can increase production of NO by upregulating iNOS by proinflammatory cytokines, whose synthesis is amplified by homocysteine. But in these conditions homocysteine induces increased expression of iNOS, and increment of O2- production by uncoupling of iNOS. [7]
Recently, it has been indicated as a potentially new mechanism involved in Hcy induced changes in cardiovascular system (CVS) which implies Toll-like receptor 4 (TLR4). TLR4 belongs to the Toll-like receptor (TLR) family, and their roles in pathogenesis of CVD are intensively studied. Namely, TLR4, that normally play a role in the innate immune response and recognize viral or bacterial antigen motifs, are expressed in almost all cells represented in CVS [8] and participate in pathogenesis of atherosclerosis [9], ischemic heart disease [10], heart failure [11] or aorta aneurysm [12]. Results of Jeremic and colleagues indicates that ablation of TLR4 in HHcy mice diminish changes induced by increased levels of Hcy such as left ventricular hypertrophy, increased oxidative stress and decreased antioxidative capacity, and mitochondrial fission [13]. Same authors provided results on the basis of which it can be concluded that TLR4 during HHcy mediate in predominance of mitochondrial fission in endothelial cells, and consequent oxidative stress, endothelial cell loss and dysfunction, increased collagen deposition, which ultimately causes hypertension [14]. On the other hand, mutation of TLR4 alleviated vascular inflammation and prevented hypertension [15]. Molecular mechanisms and signaling pathways involved in above mentioned changes in CVS induced by HHcy trough TLR4 are probably same or similar to those that exist in other cells and tissues. Intracellular domain of TLR4, Toll/interleukin-1 receptor (TIR) domain, activates several adapter proteins such as: myeloid differentiation primary response gene 88 (MyD88), Toll-Interleukin I receptor domain-containing adaptor protein (TIRAP), TIR domain-containing adaptor protein inducing interferon-b (TRIF), TRIF-related adaptor molecule (TRAM), which actually represents the first step of TLR signal transduction [16]. MyD88 further engages IL-1R-associated kinases (IRAKs) which initiate activation of nuclear factor kappa light-chain-enhancer of activated B cells (NF-κB) and production of cytokines. Another, TRIF pathway, requires TRAM, which through activation of several kinases (receptor-interacting serine/threonine-protein 1 (RIP-1) kinase and transforming growth-factor-b-activated kinase 1 (TAK-1)) activates NF-κB and mitogen-activated protein kinase (MAPK), and overall result is increment of expression of proinflammatory genes and synthesis of inflammatory cytokines. [17]
Effects of Hcy and its different forms on heart, coronary circulation and heart tissue oxidative balance were intensively investigated by Jakovljevic and Djuric. In study dealing with correlation between plasma total Hcy (tHcy) level and coronary atherosclerosis, it has been shown that tHcy was significantly higher in patients with angiographically confirmed coronary artery disease (CAD) compared to healthy control [18]. Besides that, in groups of patients with CAD, levels of tHcy more frequently exceeded the value of 15 mmol/l, which was also more common with older people, and there was positive correlation between increased tHcy level and uric acid level. In a study that examined the effects of various Hcy-related compounds (DL-Hcy, DL-Hcy Thiolactone-hydrochloride (TLHC) and L-Hcy TLHC) the authors emphasized their harmful effects on cardiac function during acute administration in an isolated rat heart [19]. During acute application of DL-Hcy TLHC simultaneous inhibition of production of the gasotransmitters (NO, H2S and CO) additionally exacerbated the effects of DL-Hcy TLHC [20]. The inhibition of CO production expressed most deleterious effects in comparison to deprivation of NO and H2S production. Furthermore, results of another investigation showed protective effects of H2S in changes induced by DL-Hcy [21]. Namely, application of DL-propargylglycine, as an inhibitor of H2S formation, decreased all cardio-dynamic parameters and increased the concentration of O2-, which was even more pronounced with the simultaneous application with DL-Hcy, which leads to the conclusion that DL-Hcy shows a lower pro-oxidative effect in the presence of H2S. It is also shown that previously mentioned Hcy-related compounds (DL-Hcy, DL-Hcy TLHC and L-Hcy TLHC) impair oxygen consumption of rat heart tissue homogenate, as well as the inclusion of the gasotransmitters NO, H2S and CO in these effects. [22]
Bearing in mind that inflammation is important factor in pathogenesis of cardiovascular diseases, a certain number of papers dealt with role of Hcy in induction of inflammation and showed that HHcy is accompanied with upregulation of several pro-inflammatory cytokines, including IL-1β, IL-6, TNF-α, MCP-1, and intracellular adhesion molecule-1. NMDA receptors are also present in almost all blood cells so that their activation may have various effects on the signaling pathways in these cells. Activation of NMDA receptors in red blood cells (RBC) induces increase in Ca2+ content and thus changes properties of RBCs such as cell volume, membrane steadiness, and capacity to transfer O2 [23].
On the other hand, Reinhart and coworkers indicated that neither activation nor inhibition has any influence on biophysical properties of RBCs, such as deformability and aggregability parameters [24]. Namely, treatment of RBCs with homocysteic acid, and combination of memantine and homocysteic acid did not change any of the observed parameters. Bearing in mind that plasma concentrations of Hcy in healthy population are below 15 μmol/l, in cases of severe HHcy, when Hcy concentration exceeds 100 μmol/l, homocysteine and homocysteic acid probably have a dominant role in the regulation of NMDA receptor activity in the blood cells [25]. The presence of the NMDA receptors in several types of immune competent cells was also confirmed, suggesting their role in regulation of immune response and inflammation. Similar to the aforementioned mechanisms in other types of cells, activation of NMDA receptors by homocysteine or homocysteic acid in cells of immune system induces Ca2+ accumulation, and consequent increase of reactive species production and oxidative stress, and activation of MAPK [26]. Activation of neutrophils causes increased expression of NMDA receptors on their membranes, so in the conditions of HHcy neutrophils generate large amounts of ROS and easier to undergo the degradation process [27]. These changes produce proinflammatory environment and enhance the production of pro-inflammatory cytokines such as TNF-α, interleukin-1β and interleukin-6 in neutrophils, monocytes or macrophages via NF-κB, ERK, and P2X7 stimulation [28]. Augmented production of pro- inflammatory cytokines and activation of immune cells activates both necrotic and apoptotic cell death. Platelets also express NMDA receptors, and results of several studies suggest their significant role in the functioning of platelets. NMDA receptor antagonists, such as MK-801 and memantine, induce inhibition on platelet aggregation and activation, while NMDA receptor agonists increase aggregation in the presence of low concentrations ADP with an increase in Ca2+ content in platelets [29]. In accordance with the above fact, it has been shown that Hcy causes an increase in whole blood platelet aggregation, as well as in production of O2-. Antagonists of NMDA receptors induced reduced increment of platelet aggregation induced by Hcy, while increased production of O2- remained unchanged, suggesting other possible mechanisms involved, independent of Ca2+ entry. [30]
As an integral component of several disorders including cardiovascular and cerebrovascular diseases, neurodegeneration, liver steatosis, hyperhomocysteinemia can result from deficiencies of vitamin cofactors (B6, B12, folic acid) required for Hcy metabolism and/or from genetic disorders of its metabolism. This review provides evidence that supports the causal role for HHcy in the development of CVD and nervous system disorders, and outlines several cellular and molecular mechanisms by which Hcy induces disorders of the function of these two organic systems. These mechanisms include oxidative stress, inflammation, ER stress, DNA hypomethylation, homocysteinylation, Hcy Thiolactone levels and mitochondrial dysfunction. In the future, further studies are much needed to provide more convincing evidence demonstrating the pathogenic role of HHcy in the progression of CVD and nervous system disorders in animal studies and clinical studies. Beside mentioned facts, it remains to be examined whether Hcy is a causative agent or marker of damage.
Funding: No funding sources.
Conflict of interest: None declared.
Ethical approval: The study was approved by the Institutional Ethics Committee of Dr RPGMC Kangra, Himachal Pradesh.
Karolczak K, Pieniazek A, Watala C. Inhibition of glutamate receptors reduces the homocysteine-induced whole blood platelet aggregation but does not affect superoxide anion generation or platelet membrane fluidization. Platelets. (2017)Jan 2;28(1):90-8. doi: 10.1080/09537104.2016.1204438 PMID: 27560773