Homozygous MTHFR C667T carriers ≤45 years old develop central retinal vein occlusion five years earlier than wild type.

PURPOSE: To assess age at 1st central retinal vein occlusion (CRVO) in carriers ≤ 45 years old of the methylenetetrahydrofolate reductase (MTHFR) C667T genotype compared to heterozygous and wild type, and to identify predictors of age at CRVO. METHODS: Retrospective cohort study consisting of 18 MTHFR TT, 23 MTHFR TC and 28 MTHFR CC participants; information regarding age, sex, age at CRVO, history of dyslipidaemia, hypertension, smoking and plasma HC measured by immunoassay were collected. RESULTS: Age at CRVO was lower in MTHFR TT than MTHFR TC and CC (32 ± 6 vs 38 ± 5 vs 37 ± 6 years, respectively, p = 0.005); plasma HC was higher in MTHFR TT than in the other genotypes [14.4 (10.8, 19.6) vs 10.4 ((8.6,12.5) vs 8.5 ((7.5,9.8) µmol/l, p = 0.0002). Smoking (cigarettes/day) independently predicted age at CRVO (p = 0.039) and plasma HC (p = 0.005); smoking status (yes/no) predicted ischemic CRVO (p = 0.01) that was more common in the MTHFR TT group (p = 0.006). CONCLUSIONS: Carriers of the MTHFR TT genotype ≤ 45 years old develop their 1st CRVO on average 5 years earlier than the MTHFR CC genotype; smoking contributes to the prematurity and severity of CRVO in MTHFR TT carriers.

Subclinical atherosclerosis in Behcet's disease and its inverse relation to azathioprine use: an updated meta-analysis.

To evaluate the intima media thickness of carotid arteries (IMT) and its clinical, laboratory and treatment correlates in Behcet's disease (BD). Systematic search of EMBASE and PubMed databases from January 2016 to October 2022; we employed random effect meta-analyses for continuous outcomes and Peto's odds ratio for rare events. The meta-analysis included 36 case control studies: the IMT was greater in BD (n = 1103) than in controls (n = 832) (p < 0.0001) with wide heterogeneity (I2 = 86.9%); a sensitivity analysis that included mean age of BD participants, gender, disease duration and activity, atherogenic index of plasma, blood pressure, C-reactive protein, ethnicity, smoking status, anti-inflammatory and immune suppressive agents, revealed that male gender, mean age of participants and azathioprine use (the latter two in inverse fashion) partly explained the heterogeneity variance (p = 0.02, p = 0.005, and p = 0.01). The IMT was greater in vascular (n = 114) than in non-vascular BD (n = 214) (p = 0.006). BD patients (n = 782) had a greater pooled prevalence of carotid plaques than controls (n = 537) (13.1% vs. 2.97%, p < 0.0001). Subclinical carotid artery atherosclerosis represents a vascular feature of BD, independently of the traditional cardiovascular risk factors. The inverse correlations between IMT, age and azathioprine use suggest that thicker carotid arteries at disease onset eventually regress with immune suppressive treatment: this assumption needs verification on adequately designed clinical trials.

Earlier onset of peripheral arterial thrombosis in homozygous MTHFR C677T carriers than in other MTHFR genotypes: a cohort study.

To investigate whether age at first presentation of pure peripheral arterial thrombosis (PAT) in lower and upper limbs and in the splanchnic circulation occurs earlier in carriers of the methylenetetrahydrofolate reductase (MTHFR) T677T genotype compared to the heterozygous and wild type and to identify predictors of a possible earlier onset. Retrospective cohort study on 27 MTHFR TT, 29 MTHFR TC and 29 MTHFR CC participants; data regarding age, sex, age at PAT, clinical history (dyslipidaemia, hypertension, smoking, obesity) and homocysteine (HC) measured by immunoassay were collected. Age at PAT was lower in MTHFR TT than MTHFR TC and CC (43 ± 9 vs 47 ± 9 vs 51 ± 4 years, respectively, p = 0.02); plasma HC was higher in MTHFR TT than in the other groups (25 ± 19 vs 12.7 ± 6.7 vs 11.3 ± 3.3 µmol/l, respectively, p < 0.001) while the activated partial thromboplastin ratio (aPTTr) was lower in MTHFR TT than in other genotypes (0.90 ± 0.10 vs 0.97 ± 0.12 vs 0.97 ± 0.08 µmol/L p < 0.001). Among categorical variables, MTHFR TT and dyslipidaemia independently predicted age at AT (p = 0.01 & p = 0.03, respectively) whereas among the continuous variables HC independently predicted age at PAT (p = 0.02) as well as the aPTTr (p = 0.001); smoking predicted lower limb PAT (p = 0.005). MTHFR TT carriers develop their first PAT an average of 4 and 8 years earlier than MTHF CT and CC genotypes; MTHFR TT, dyslipidaemia and plasma HC contribute to the prematurity of the PAT while the interplay between elevated HC and smoking may affect type of arterial district occlusion.

Juvenile patients with the homozygous MTHFR C677T genotype develop ischemic stroke 5 years earlier than wild type.

To compare age at 1st ischaemic stroke (IS) in a cohort of juvenile (< 46 years of age) IS patients evaluated for the rs1801133 polymorphism (C → T677) of the methylene tetrahydrofolate reductase (MTHFR) gene; to identify predictors of age at IS and of type of cerebral vessel involvement, small vessel disease (SVD) vs large vessel disease (LVD) responsible for the IS; to evaluate possible associations between other clinical and laboratory variables. Retrospective cohort study on 82 MTHFR TT, 54 MTHFR TC and 34 MTHFR CC participants; data regarding age, sex, age at IS, history of dyslipidaemia, hypertension, smoking, migraine and homocysteine (HC) as well as neuroimaging were collected. Age at IS was lower in MTHFR TT than MTHFR TC and CC (35 ± 4 vs 38 ± 0 vs 40 ± 3 years, respectively, p = 0.002); plasma HC (median, interquartile range) was higher in MTHFR TT than in the other groups [16.7 (11.8, 28.6) vs 11.4 (8.2, 16.1) vs 9.8 (7.9, 1.3) respectively, p < 0.0001)] and was higher in SVD than LVD [17.4 (12.4, 32.5) vs  11.4 (8.8, 16.4) p < 0.0001]. MTHFR TT independently predicted age at IS (p = 0.0008) alongside smoking both as a categorical (p = 0.003) or continuous variable (p = 0.02), whereas HC independently predicted SVD as categorical (p = 0.01) and continuous variable (p < 0.0001). Smoking positively predicted plasma HC (p = 0.005) and negatively the activated partial thromboplastin ratio (aPTTr) (p = 0.02). Juvenile IS carriers of the MTHFR TT genotype develop their 1st occlusion on average 5 years earlier compared to the CC genotype; smoking contributes to this prematurity adversely affecting plasma HC and coagulation whereas plasma HC predicts IS secondary to SVD. Public health campaigns against smoking should highlight the prematurity of IS in the juvenile population.

Lipid Peroxidation-Derived Aldehydes, 4-Hydroxynonenal and Malondialdehyde in Aging-Related Disorders.

Among the various mechanisms involved in aging, it was proposed long ago that a prominent role is played by oxidative stress. A major way by which the latter can provoke structural damage to biological macromolecules, such as DNA, lipids, and proteins, is by fueling the peroxidation of membrane lipids, leading to the production of several reactive aldehydes. Lipid peroxidation-derived aldehydes can not only modify biological macromolecules, by forming covalent electrophilic addition products with them, but also act as second messengers of oxidative stress, having relatively extended lifespans. Their effects might be further enhanced with aging, as their concentrations in cells and biological fluids increase with age. Since the involvement and the role of lipid peroxidation-derived aldehydes, particularly of 4-hydroxynonenal (HNE), in neurodegenerations, inflammation, and cancer, has been discussed in several excellent recent reviews, in the present one we focus on the involvement of reactive aldehydes in other age-related disorders: osteopenia, sarcopenia, immunosenescence and myelodysplastic syndromes. In these aging-related disorders, characterized by increases of oxidative stress, both HNE and malondialdehyde (MDA) play important pathogenic roles. These aldehydes, and HNE in particular, can form adducts with circulating or cellular proteins of critical functional importance, such as the proteins involved in apoptosis in muscle cells, thus leading to their functional decay and acceleration of their molecular turnover and functionality. We suggest that a major fraction of the toxic effects observed in age-related disorders could depend on the formation of aldehyde-protein adducts. New redox proteomic approaches, pinpointing the modifications of distinct cell proteins by the aldehydes generated in the course of oxidative stress, should be extended to these age-associated disorders, to pave the way to targeted therapeutic strategies, aiming to alleviate the burden of morbidity and mortality associated with these disturbances.

DNA damage by lipid peroxidation products: implications in cancer, inflammation and autoimmunity.

Oxidative stress and lipid peroxidation (LPO) induced by inflammation, excess metal storage and excess caloric intake cause generalized DNA damage, producing genotoxic and mutagenic effects. The consequent deregulation of cell homeostasis is implicated in the pathogenesis of a number of malignancies and degenerative diseases. Reactive aldehydes produced by LPO, such as malondialdehyde, acrolein, crotonaldehyde and 4-hydroxy-2-nonenal, react with DNA bases, generating promutagenic exocyclic DNA adducts, which likely contribute to the mutagenic and carcinogenic effects associated with oxidative stress-induced LPO. However, reactive aldehydes, when added to tumor cells, can exert an anticancerous effect. They act, analogously to other chemotherapeutic drugs, by forming DNA adducts and, in this way, they drive the tumor cells toward apoptosis. The aldehyde-DNA adducts, which can be observed during inflammation, play an important role by inducing epigenetic changes which, in turn, can modulate the inflammatory process. The pathogenic role of the adducts formed by the products of LPO with biological macromolecules in the breaking of immunological tolerance to self antigens and in the development of autoimmunity has been supported by a wealth of evidence. The instrumental role of the adducts of reactive LPO products with self protein antigens in the sensitization of autoreactive cells to the respective unmodified proteins and in the intermolecular spreading of the autoimmune responses to aldehyde-modified and native DNA is well documented. In contrast, further investigation is required in order to establish whether the formation of adducts of LPO products with DNA might incite substantial immune responsivity and might be instrumental for the spreading of the immunological responses from aldehyde-modified DNA to native DNA and similarly modified, unmodified and/or structurally analogous self protein antigens, thus leading to autoimmunity.

Mitochondrial Dysfunction in Cancer and Neurodegenerative Diseases: Spotlight on Fatty Acid Oxidation and Lipoperoxidation Products.

In several human diseases, such as cancer and neurodegenerative diseases, the levels of reactive oxygen species (ROS), produced mainly by mitochondrial oxidative phosphorylation, is increased. In cancer cells, the increase of ROS production has been associated with mtDNA mutations that, in turn, seem to be functional in the alterations of the bioenergetics and the biosynthetic state of cancer cells. Moreover, ROS overproduction can enhance the peroxidation of fatty acids in mitochondrial membranes. In particular, the peroxidation of mitochondrial phospholipid cardiolipin leads to the formation of reactive aldehydes, such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), which are able to react with proteins and DNA. Covalent modifications of mitochondrial proteins by the products of lipid peroxidation (LPO) in the course of oxidative cell stress are involved in the mitochondrial dysfunctions observed in cancer and neurodegenerative diseases. Such modifications appear to affect negatively mitochondrial integrity and function, in particular energy metabolism, adenosine triphosphate (ATP) production, antioxidant defenses and stress responses. In neurodegenerative diseases, indirect confirmation for the pathogenetic relevance of LPO-dependent modifications of mitochondrial proteins comes from the disease phenotypes associated with their genetic alterations.

Role of 4-hydroxynonenal-protein adducts in human diseases.

SIGNIFICANCE: Oxidative stress provokes the peroxidation of polyunsaturated fatty acids in cellular membranes, leading to the formation of aldheydes that, due to their high chemical reactivity, are considered to act as second messengers of oxidative stress. Among the aldehydes formed during lipid peroxidation (LPO), 4-hydroxy-2-nonenal (HNE) is produced at a high level and easily reacts with both low-molecular-weight compounds and macromolecules, such as proteins and DNA. In particular, HNE-protein adducts have been extensively investigated in diseases characterized by the pathogenic contribution of oxidative stress, such as cancer, neurodegenerative, chronic inflammatory, and autoimmune diseases. RECENT ADVANCES: In this review, we describe and discuss recent insights regarding the role played by covalent adducts of HNE with proteins in the development and evolution of those among the earlier mentioned disease conditions in which the functional consequences of their formation have been characterized. CRITICAL ISSUES: Results obtained in recent years have shown that the generation of HNE-protein adducts can play important pathogenic roles in several diseases. However, in some cases, the generation of HNE-protein adducts can represent a contrast to the progression of disease or can promote adaptive cell responses, demonstrating that HNE is not only a toxic product of LPO but also a regulatory molecule that is involved in several biochemical pathways. FUTURE DIRECTIONS: In the next few years, the refinement of proteomical techniques, allowing the individuation of novel cellular targets of HNE, will lead to a better understanding the role of HNE in human diseases.

Nitroxyl (HNO) for treatment of acute heart failure.

The loss of contractile function is a hallmark of heart failure. Although increasing intracellular Ca(2+) is a possible strategy for improving contraction, current inotropic agents that achieve this by raising intracellular cAMP levels, such as ß-agonists and phosphodiesterase inhibitors, are generally deleterious when administered as long-term therapy due to arrhythmia and myocardial damage. Nitroxyl donors have been shown to improve cardiac function in normal and failing dogs, and in isolated cardiomyocytes they increase fractional shortening and Ca(2+) transients, independently from cAMP/PKA or cGMP/PKG signaling. Instead, nitroxyl targets cysteines in the EC-coupling machinery and myofilament proteins, reversibly modifying them to enhance Ca(2+) handling and myofilament Ca(2+) sensitivity. Phase I-IIa trials with CXL-1020, a novel pure HNO donor, reported declines in left and right heart filling pressures and systemic vascular resistance, and increased cardiac output and stroke volume index. These findings support the concept of nitroxyl donors as attractive agents for the treatment of acute decompensated heart failure.

Interaction of aldehydes derived from lipid peroxidation and membrane proteins.

A great variety of compounds are formed during lipid peroxidation of polyunsaturated fatty acids of membrane phospholipids. Among them, bioactive aldehydes, such as 4-hydroxyalkenals, malondialdehyde (MDA) and acrolein, have received particular attention since they have been considered as toxic messengers that can propagate and amplify oxidative injury. In the 4-hydroxyalkenal class, 4-hydroxy-2-nonenal (HNE) is the most intensively studied aldehyde, in relation not only to its toxic function, but also to its physiological role. Indeed, HNE can be found at low concentrations in human tissues and plasma and participates in the control of biological processes, such as signal transduction, cell proliferation, and differentiation. Moreover, at low doses, HNE exerts an anti-cancer effect, by inhibiting cell proliferation, angiogenesis, cell adhesion and by inducing differentiation and/or apoptosis in various tumor cell lines. It is very likely that a substantial fraction of the effects observed in cellular responses, induced by HNE and related aldehydes, be mediated by their interaction with proteins, resulting in the formation of covalent adducts or in the modulation of their expression and/or activity. In this review we focus on membrane proteins affected by lipid peroxidation-derived aldehydes, under physiological and pathological conditions.

Exposure of HL-60 human leukaemic cells to 4-hydroxynonenal promotes the formation of adduct(s) with alpha-enolase devoid of plasminogen binding activity.

HNE (4-hydroxynonenal), the major product of lipoperoxidation, easily reacts with proteins through adduct formation between its three main functional groups and lysyl, histidyl and cysteinyl residues of proteins. HNE is considered to be an ultimate mediator of toxic effects elicited by oxidative stress. It can be detected in several patho-physiological conditions, in which it affects cellular processes by addition to functional proteins. We demonstrated in the present study, by MS and confirmed by immunoblotting experiments, the formation of HNE-alpha-enolase adduct(s) in HL-60 human leukaemic cells. Alpha-enolase is a multifunctional protein that acts as a glycolytic enzyme, transcription factor [MBP-1 (c-myc binding protein-1)] and plasminogen receptor. HNE did not affect alpha-enolase enzymatic activity, expression or intracellular localization, and did not change the expression and localization of MBP-1 either. Confocal and electronic microscopy results confirmed the plasma membrane, cytosolic and nuclear localization of alpha-enolase in HL-60 cells and demonstrated that HNE was colocalized with alpha-enolase at the surface of cells early after its addition. HNE caused a dose- and time-dependent reduction of the binding of plasminogen to alpha-enolase. As a consequence, HNE reduced adhesion of HL-60 cells to HUVECs (human umbilical vein endothelial cells). These results could suggest a new role for HNE in the control of tumour growth and invasion.

A single chondroitin 6-sulfate oligosaccharide unit at Ser-2730 of human thyroglobulin enhances hormone formation and limits proteolytic accessibility at the carboxyl terminus. Potential insights into thyroid homeostasis and autoimmunity.

We localized the site of type D (chondroitin 6-sulfate) oligosaccharide unit addition to human thyroglobulin (hTg). hTg was chromatographically separated into chondroitin 6-sulfate-containing (hTg-CS) and chondroitin 6-sulfate-devoid (hTg-CS0) molecules on the basis of their D-glucuronic acid content. In an ample number of hTg preparations, the fraction of hTg-CS in total hTg ranged from 32.0 to 71.6%. By exploiting the electrophoretic mobility shift and metachromasia conferred by chondroitin 6-sulfate upon the products of limited proteolysis of hTg, chondroitin 6-sulfate was first restricted to a carboxyl-terminal region, starting at residue 2514. A single chondroitin 6-sulfate-containing nonapeptide was isolated in pure form from the products of digestion of hTg with endoproteinase Glu-C, and its sequence was determined as LTAGXGLRE (residues 2726-2734, X being Ser2730 linked to the oligosaccharide chain). In an in vitro assay of enzymatic iodination, hTg-CS produced higher yields of 3,5,5 '-triiodothyronine (T3) (171%) and 3,5,3',5'-tetraiodothyronine (T4) (134%) than hTg-CS0. Unfractionated hTg behaved as hTg-CS. Thus, chondroitin 6-sulfate addition to a subset of hTg molecules enhanced the overall level of T4 and, in particular, T3 formation. Furthermore, the chondroitin 6-sulfate oligosaccharide unit of hTg-CS protected peptide bond Lys2714-Gly2715 from proteolysis, during the limited digestion of hTg-CS with trypsin. These findings provide insights into the molecular mechanism of regulation of the hormonogenic efficiency and of the T4/T3 ratio in hTg. The potential implications in the ability of hTg to function as an autoantigen and into the pathogenesis of thyroidal and extra-thyroidal manifestations of autoimmune thyroid disease are discussed.