Anti-angiogenic effects of homocysteine on cultured endothelial cells

High levels of homocysteine induce a sustained injury on arterial endothelial cells which accelerates the development of thrombosis and atherosclerosis. Some of the described effects of homocysteine on endothelial cells are features shared with an anti-angiogenic response. Therefore, we studied the effects of homocysteine on key steps of angiogenesis using bovine aorta endothelial cells as a model. Homocysteine decreased proliferation and induced differentiation. Furthermore, 5 mM homocysteine produced strong inhibitions of matrix metalloproteinase-2 and urokinase, two proteolytic activities that play a key role in extracellular matrix re-modeling, and decreased migration and invasion, other two key steps of angiogenesis. This study demonstrates that homocysteine can inhibit several steps of the angiogenic process.     

Homocysteine inhibits the proliferation and invasive potential of HT-1080 human fibrosarcoma cells

The impairment of homocysteine metabolism has been related to several disorders and diseases. Recently, homocysteine has been shown to inhibit key steps of angiogenesis, including endothelial cell proliferation, invasion, and remodeling of the extracellular matrix. Since these are also key steps in tumor invasion and metastasis, it can be hypothesized that homocysteine can also interfere in these processes. Therefore, we studied the effects of homocysteine on tumor proliferation and invasion, as well as on urokinase, a key extracellular matrix-degrading protease, using a model human tumor cell line. This study demonstrates that, in fact, homocysteine inhibits HT-1080 proliferation and invasion, and is a potent inhibitor of tumor cell urokinase expression.     

Homocysteine is a potent inhibitor of human tumor cell gelatinases

Extracellular matrix-degrading gelatinases are mainly involved in tumor invasion and metastasis. Previous experimental data from our group and others suggested that homocysteine could have a potential modulatory role on the proteolytic balance at the extracellular matrix. Therefore, we studied the effects of homocysteine on extracellular matrix-degrading proteases using model human tumor cell lines and a combination of in vitro fluorogenic assay and zymographic techniques. Homocysteine is shown to be the thiol compound with the most potent inhibitory activity on matrix metalloproteinase 9. Zymographies reveal that matrix metalloproteinase 2 is, at least, as sensitive to inhibition by homocysteine as matrix metalloproteinase 9 is. This study opens new ways to the potential pharmacological use of thiol compounds.     

Homocysteine pre-treatment increases redox capacity in both endothelial and tumor cells

Objective: We studied the modulatory effects of homocysteine pre-treatment on the disulfide reduction capacity of tumor and endothelial cells.

Methods: Human MDA-MB-231 breast carcinoma and bovine aorta endothelial cells were pre-treated for 1–24 hours with 0.5–5 mM homocysteine or homocysteine thiolactone. After washing to eliminate any rest of homocysteine or homocysteine thiolactone, cell redox capacity was determined by using a method for measuring disulfide reduction.

Results: Homocysteine pre-treatments for 1–4 hours at a concentration of 0.5–5 mM increase the disulfide reduction capacity of both tumor and endothelial cells. This effect cannot be fully mimicked by either cysteine or homocysteine thiolactone pre-treatments of tumor cells.

Discussion: Taken together, our data suggest that homocysteine can behave as an anti-oxidant agent by increasing the anti-oxidant capacity of tumor and endothelial cells.     

Major metabolic homocysteine-derivative, homocysteine thiolactone, exerts changes in pancreatic beta-cell glucose-sensing, cellular signal transduction and integrity

Homocysteine can be converted to its reactive thioester, homocysteine thiolactone. Cytotoxic properties of these amino thiols have been attributed to protein homocysteinylation, increased oxidative stress, DNA damage and apoptosis. This study used pancreatic BRIN-BD11 beta-cells to examine functional defects caused by acute and long-term exposure to homocysteine thiolactone in comparison with homocysteine. Acute and long-term exposure to both agents caused concentration-dependent inhibitions of glucose-induced insulin secretion while impairing the insulin-secretory responses to alanine, KCl, elevated Ca(2+), forskolin and PMA. Acute exposures also caused significant reduction in the amplitude of KCl-induced membrane depolarisation but no effects on changes of intracellular Ca(2+) induced by alanine or KCl. Cellular insulin content and DNA damage were not altered following culture, however, there were early signs of apoptosis consistent with impaired cellular integrity. In conclusion, exposure to homocysteine thiolactone, like homocysteine, induced beta-cell dysfunction and demise by mechanisms independent of changes in membrane potential and [Ca(2+)](i).     

Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels.

Homocysteine thiolactone, a cyclic thioester, is synthesized by certain aminoacyl-tRNA synthetases in editing or proofreading reactions that prevent translational incorporation of homocysteine into proteins. Although homocysteine thiolactone is expected to acylate amino groups in proteins, virtually nothing is known regarding reactivity of the thiolactone. Here it is shown that reactions of the thiolactone with protein lysine residues were robust under physiological conditions. In human serum incubated with homocysteine thiolactone, protein homocysteinylation was a major reaction that could be observed with as little as 10 nM thiolactone. Individual proteins were homocysteinylated at rates proportional to their lysine contents. Homocysteinylation led to protein damage, manifested as multimerization and precipitation of extensively modified proteins. Model enzymes, such as methionyl-tRNA synthetase and trypsin, were inactivated by homocysteinylation. Metabolic conversion of homocysteine to the thiolactone, protein homocysteinylation, and resulting protein damage may underlie involvement of Hcy in the pathology of vascular disease.-Jakubowski, H. Protein homocysteinylation: possible mechanism underlying pathological consequences of elevated homocysteine levels.     

Homocysteine Thiolactone and Human Cholinesterases

1. The cholinergic system is important in cognition and behavior as well as in the function of the cerebral vasculature. 2. Hyperhomocysteinemia is a risk factor for development of both dementia and cerebrovascular disease. 3. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are serine hydrolase enzymes that catalyze the hydrolysis of the neurotransmitter acetylcholine, a key process in the regulation of the cholinergic system. 4. It has been hypothesized that the deleterious effects of elevated homocysteine may, in part, be due to its actions on cholinesterases. 5. To further test this hypothesis, homocysteine and a number of its metabolites and analogues were examined for effects on the activity of human cholinesterases. 6. Homocysteine itself did not have any measurable effect on the activity of these enzymes. 7. Homocysteine thiolactone, the cyclic metabolite of homocysteine, slowly and irreversibly inhibited the activity of human AChE. 8. Conversely, this metabolite and some of its analogues significantly enhanced the activity of human BuChE. 9. Structure–activity studies indicated that the unprotonated amino group of homocysteine thiolactone and related compounds represents the essential feature for activation of BuChE, whereas the thioester linkage appears to be responsible for the slow AChE inactivation. 10. It is concluded that hyperhomocysteinemia may exert its adverse effects, in part, through the metabolite of homocysteine, homocysteine thiolactone, which is capable of altering the activity of human cholinesterases, the most pronounced effect being BuChE activation.     

Homocysteine and other structurally-diverse amino thiols can alter pancreatic beta cell function without evoking cellular damage

Homocysteine and related amino thiols, homocysteic acid, cysteic acid, homocysteine sulphinic acid and cysteine sulphinic acid have been labelled as neurotoxins. Homocysteine thiolactone, a metabolic derivative of homocysteine, is cytotoxic to endothelial cells and other cell lineages. Since pancreatic beta cells share many phenotypic similarities with neuronal cells, the present study uses clonal pancreatic BRIN-BD11 cells to investigate possible detrimental effects of these amino thiols on insulin secretion and pancreatic beta cell function. Insulin secretion was concentration-dependently inhibited at both basal (1.1 mM) and stimulatory (16.7 mM) glucose by homocysteine, homocysteine thiolactone and homocysteine sulphinic acid. Cysteic acid concentration-dependently inhibited insulin secretion at 16.7 mM glucose. Cell viability was not compromised by any of the amino thiols. Insulin secretory responses to alanine were inhibited by homocysteine, homocysteine thiolactone, homocysteic acid and cysteic acid. Insulin secretion in the presence of elevated Ca(2+) and forskolin were lowered by all amino thiols, except homocysteic acid. The secretory responsiveness to PMA, GLP-1 and KCl were only impaired in the presence of homocysteine and homocysteine thiolactone. These findings indicate that homocysteine, homocysteine thiolactone and, to a lesser extent, other amino thiols cause dysfunctional insulin secretion from pancreatic beta cells.     

Mechanisms of homocysteine toxicity in humans

Summary. Homocysteine, a non-protein amino acid, is an important risk factor for ischemic heart disease and stroke in humans. This review provides an overview of homocysteine influence on endothelium function as well as on protein metabolism with a special respect to posttranslational modification of protein with homocysteine thiolactone. Homocysteine is a pro-thrombotic factor, vasodilation impairing agent, pro-inflammatory factor and endoplasmatic reticulum-stress inducer. Incorporation of Hcy into protein via disulfide or amide linkages (S-homocysteinylation or N-homocysteinylation) affects protein structure and function. Protein N-homocysteinylation causes cellular toxicity and elicits autoimmune response, which may contribute to atherogenesis. Present address: Department of Biochemistry and Biotechnology, Agricultural University, 60637 Poznań, Poland     

Effects of ursolic acid on different steps of the angiogenic process

Ursolic acid is a triterpenoid with pleiotropic biological effects. In this report, we study the effects of ursolic acid on different key steps of angiogenesis. Our results show that ursolic acid is able to inhibit key steps of angiogenesis in vitro, including endothelial cell proliferation, migration, and differentiation. At the same time, it seems to stimulate other key steps of angiogenesis, such as extracellular matrix degradation by MMP-2 and urokinase. Although ursolic acid can inhibit in vivo angiogenesis in the CAM assay, the different signs of the effects it causes on different steps of angiogenesis force one to be cautious concerning its anti-angiogenic potential.     

Proofreading in vivo: editing of homocysteine by methionyl-tRNA synthetase in the yeast Saccharomyces cerevisiae.

Homocysteine thiolactone is a product of an error-editing reaction, catalyzed by Escherichia coli methionyl-tRNA synthetase, which prevents incorporation of homocysteine into tRNA and protein, both in vitro and in vivo. Here, the thiolactone is also shown to occur in cultures of the yeast Saccharomyces cerevisiae. In yeast, the thiolactone is made from homocysteine in a reaction catalyzed by methionyl-tRNA synthetase. One molecule of homocysteine is edited as thiolactone per 500 molecules of methionine incorporated into protein. Homocysteine, added exogenously to the medium or overproduced by some yeast mutants, is detrimental to cell growth. The cost of homocysteine editing in yeast is minimized by the presence of a pathway leading from homocysteine to cysteine, which keeps intracellular homocysteine at low levels. These results not only directly demonstrate that editing of errors in amino acid selection by methionyl-tRNA synthetase operates in vivo in yeast but also establish the importance of proofreading mechanisms in a eukaryotic organism.     

The effect of homocysteine thiolactone and its alpha methylated derivative on bone matrix in the mouse

Summary Homocysteine (HC) is a radiation protector but toxic to bone. Its derivative homocysteine thiolactone (HCTL) and the alpha-alkylated analogue (A-methyl-HCTL) was fed to mice for a period of six weeks in a daily dose of 50 mg/kg body weight. Parameters for bone matrix as collagen content, acid solubility of bone collagen, urinary bone collagen cross links (pyridinolines) and urinary acid glycosaminoglycans were determined. Urinary acid glycosaminoglycans were significantly reduced in the HCTL treated group but not in the alpha-methyl-homocysteine thiolactone (A-methyl-HCTL) group (controls: 45 ± 7 mg/mmol creatinine, homocysteine thiolactone 38 ± 5 mg/mmol creatinine, A-methyl HCTL 45 ± 6 mg/mmol creatinine).No differences were found for the parameters of bone collagen between the groups. The potent radiation protecting methylated derivative therefore did not change bone matrix and should be a candidate for further toxicological studies.     

A versatile equation to describe reversible enzyme inhibition and activation kinetics: Modeling β-galactosidase and butyrylcholinesterase

Current treatments for Alzheimer's disease involve inhibiting cholinesterases. Conversely, cholinesterase stimulation may be deleterious. Homocysteine is a known risk factor for Alzheimer's and vascular diseases and its active metabolite, homocysteine thiolactone, stimulates butyrylcholinesterase. Considering the opposing effects on butyrylcholinesterase of homocysteine thiolactone and cholinesterase inhibitors, understanding how these molecules alter this enzyme may provide new insights in the management of dementia. Butyrylcholinesterase does not strictly adhere to Michaelis–Menten parameters since, at higher substrate concentrations, enzyme activation occurs. The substrate activation equation for butyrylcholinesterase does not describe the effects of inhibitors or non-substrate activators. To address this, global data fitting was used to generate a flexible equation based on Michaelis–Menten principles. This methodology was first tested to model complexities encountered in inhibition by imidazole of β-galactosidase, an enzyme that obeys Michaelis–Menten kinetics. The resulting equation was sufficiently flexible to permit expansion for modeling activation or inhibition of butyrylcholinesterase, while accounting for substrate activation of this enzyme. This versatile equation suggests that both the inhibitor and non-substrate activator examined here have little effect on the substrate-activated form of butyrylcholinesterase. Given that butyrylcholinesterase inhibition can antagonize stimulation of this enzyme by homocysteine thiolactone, cholinesterase inhibition may have a role in treating Alzheimer and vascular diseases related to hyperhomocysteinemia.     

Homocysteine metabolism and the oxidative modification of proteins and lipids

Altered homocysteine metabolism is implicated as a pathogenic factor in atherogenesis, neoplasia, and aging. Hereditary enzymatic deficiencies and nutritional deficiencies of folate, pyridoxine, or cobalamin are associated with elevated blood homocysteine, accelerated atherosclerosis, and manifestations of aging. The failure of malignant cells to metabolize homocysteine thiolactone to sulfate is attributed to deficiency of thioretinaco, a complex containing cobalamin, homocysteine thiolactone, and retinoic acid. The sulfhydryl group of homocysteine is believed to act catalytically with ferric or cupric ions in a mixed function oxidation system to generate hydrogen peroxide, oxygen radicals, and homocysteinyl radicals. These reactive species may interact with the active site of enzyme protein to cause inactivation of catalytic activity. Homocysteine thiolactone is oxidized to sulfae by a process involving ascorbate, thioretinamide, and superoxide, under the control of thyroxine and growth hormone. Thioretinaco is believed to be the active site of adenosine triphosphate (ATP) binding in oxidative phosphorylation with the participation of oxygen, ascorbate, proton gradient, and electron transport. Depletion of thioretinaco from mitochondrial and microsomal membranes may be associated with increased formation and release of radical oxygen species within neoplastic and senescent cells. Specific proposals are made for investigating the importance of homocysteine metabolism in the oxidative modification of proteins and lipids.     

Existence of Molten Globule State in Homocysteine-Induced Protein Covalent Modifications

Homocysteine thiolactone is a toxic metabolite produced from homocysteine by amino-acyl t-RNA synthetase in error editing reaction. The basic cause of toxicity of homocysteine thiolactone is believed to be due to the adduct formation with lysine residues (known as protein N-homocysteinylation) leading to protein aggregation and loss of enzyme function. There was no data available until now that showed the effect of homocysteine thiolactone on the native state structural changes that led to aggregate formation. In the present study we have investigated the time dependent structural changes due to homocysteine thiolactone induced modifications on three different proteins having different physico-chemical properties (cytochrome-c, lysozyme and alpha lactalbumin). We discovered that N-homocysteinylation leads to the formation of molten globule state—an important protein folding intermediate in the protein folding pathway. We also found that the formation of the molten globule state might be responsible for the appearance of aggregate formation. The study indicates the importance of protein folding intermediate state in eliciting the homocysteine thiolactone toxicity.     

Calcium-dependent human serum homocysteine thiolactone hydrolase. A protective mechanism against protein N-homocysteinylation

Homocysteine thiolactone is formed in all cell types studied thus far as a result of editing reactions of some aminoacyl-tRNA synthetases. Because inadvertent reactions of thiolactone with proteins are potentially harmful, the ability to detoxify homocysteine thiolactone is essential for biological integrity. This work shows that a single specific enzyme, present in mammalian but not in avian sera, hydrolyzes thiolactone to homocysteine. Human serum thiolactonase, a 45-kDa protein component of high density lipoprotein, requires calcium for activity and stability and is inhibited by isoleucine and penicillamine. Substrate specificity studies suggest that homocysteine thiolactone is a likely natural substrate of this enzyme. However, thiolactonase also hydrolyzes non-natural substrates, such as phenyl acetate, p-nitrophenyl acetate, and the organophospate paraoxon. N-terminal amino acid sequence of pure thiolactonase is identical with that of human paraoxonase. These and other data indicate that paraoxonase, an organophosphate-detoxifying enzyme whose natural substrate and function remained unknown up to now, is in fact homocysteine thiolactonase. By detoxifying homocysteine thiolactone, the thiolactonase/paraoxonase would protect proteins against homocysteinylation, a potential contributing factor to atherosclerosis.     

Hydrolysis of homocysteine thiolactone results in the formation of Protein-Cys-S-S-homocysteinylation

An increased level of homocysteine, a reactive thiol amino acid, is associated with several complex disorders and is an independent risk factor for cardiovascular disease. A majority (>80%) of circulating homocysteine is protein bound. Homocysteine exclusively binds to protein cysteine residues via thiol disulfide exchange reaction, the mechanism of which has been reported. In contrast, homocysteine thiolactone, the cyclic thioester of homocysteine, is believed to exclusively bind to the primary amine group of lysine residue leading to N-homocysteinylation of proteins and hence studies on binding of homocysteine thiolactone to proteins thus far have only focused on N-homocysteinylation. Although it is known that homocysteine thiolactone can hydrolyze to homocysteine at physiological pH, surprisingly the extent of S-homocysteinylation during the exposure of homocysteine thiolactone with proteins has never been looked into. In this study, we clearly show that the hydrolysis of homocysteine thiolactone is pH dependent, and at physiological pH, 1 mM homocysteine thiolactone is hydrolysed to ~0.71 mM homocysteine within 24 h. Using albumin, we also show that incubation of HTL with albumin leads to a greater proportion of S-homocysteinylation (0.41 mol/mol of albumin) than N-homocysteinylation (0.14 mol/mol of albumin). S-homocysteinylation at Cys³⁴ of HSA on treatment with homocysteine thiolactone was confirmed using LC-MS. Further, contrary to earlier reports, our results indicate that there is no cross talk between the cysteine attached to Cys³⁴ of albumin and homocysteine attached to lysine residues.     

The disturbance of hemostasis induced by hyperhomocysteinemia; the role of antioxidants

Elevated concentration of homocysteine (Hcy) in human tissues, definied as hyperhomocysteinemia has been correlated with some diseases, such as cardiovascular, neurodegenerative, and kidney disorders. Homocysteine occurs in human blood plasma in several forms, including the most reactive one, the homocysteine thiolactone (HTL) - a cyclic thioester, which represents up to 0.29% of total plasma Hcy. In the article, the effects of hyperhomocysteinemia on the complex process of hemostasis, which regulates the flowing properties of blood, are described. Possible interactions of homocysteine and its different derivatives, including homocysteine thiolactone, with the major components of hemostasis such as endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen, are also discussed. Modifications of hemostatic proteins (N-homocysteinylation or S-homocysteinylation) induced by Hcy or its thiolactone seem to be the main cause of homocysteine biotoxicity in hemostatic abnormalities. It is suggested that Hcy and HTL may also act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. We also discuss the role of phenolic antioxidants in hyperhomocysteinemia -induced changes in hemostasis.     

Growth promotion by homocysteine thiolactone and its alpha-alkylated derivative — A role for excess growth in homocystinurias?

Summary It is controversial whether homocysteic acid or other homocysteine derivatives show growth promoting effects. In a clonogenic assay we could show that homocysteine thiolactone and its alpha alkylated derivative increased colony formation significantly. Our work favorizes previous observations showing growth promoting activity of homocysteine derivatives and encourages further studies on that subject with implications for growth in physioogy and under pathological conditions.     

Purification and kinetic properties of rabbit liver paraoxonase 1

Paraoxonase 1 (PON1) is synthesized in the liver and secreted into the blood, where it is associated exclusively with HDL. In this study, rabbit liver PON1 enzyme was purified to homogeneity using a new purification approach, and the kinetic properties of the enzyme were investigated using phenyl acetate and homocysteine thiolactone as substrates. Rabbit liver PON1 was purified through the preparation of liver microsomal fraction, Sephacryl S300 HR gel filtration chromatography, DEAE Trisacryl M ion-exchange chromatography and hydroxyapatite chromatography steps. Using this method, rabbit liver PON1 was purified 576 times with a specific activity of 2726 U/mg protein. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed the obtained enzyme as a single protein band close to 40 kDa. The Km of the this enzyme was found as 0.55 ± 0.024 mM for phenyl acetate and 17.31 ± 1.2 mM for homocysteine thiolactone. In this study, a new approach was used to purify PON1 enzyme from rabbit liver and for the first time in the literature, its kinetics was studied with homocysteine thiolactone as substrate.