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The inability to efficiently metabolize homocysteine (Hcy) due to nutritional and genetic deficiencies, leads to hyperhomocysteinemia (HHcy) and endothelial dysfunction, a hallmark of atherosclerosis which underpins cardiovascular disease (CVD). PHF8 is a histone demethylase that demethylates H4K20me1, which affects the mammalian target of rapamycin (mTOR) signaling and autophagy, processes that play important roles in CVD. PHF8 is regulated by microRNA (miR) such as miR‐22‐3p and miR‐1229‐3p. Biochemically, HHcy is characterized by elevated levels of Hcy, Hcy‐thiolactone and N‐Hcy‐protein. Here, we examined the effects of these metabolites on miR‐22‐3p, miR‐1229‐3p, and their target PHF8, as well as on the downstream consequences of these effects on H4K20me1, mTOR‐, and autophagy‐related proteins and mRNAs expression in human umbilical vein endothelial cells (HUVEC). We found that treatments with N‐Hcy‐protein, Hcy‐thiolactone, or Hcy upregulated miR‐22‐3p and miR‐1229‐3p, attenuated PHF8 expression, upregulated H4K20me1, mTOR, and phospho‐mTOR. Autophagy‐related proteins (BECN1, ATG5, ATG7, lipidated LC3‐II, and LC3‐II/LC3‐I ratio) were significantly downregulated by at least one of these metabolites. We also found similar changes in the expression of miR‐22‐3p, Phf8, mTOR‐ and autophagy‐related proteins/mRNAs in vivo in hearts of Cbs−/− mice, which show severe HHcy and endothelial dysfunction. Treatments with inhibitors of miR‐22‐3p or miR‐1229‐3p abrogated the effects of Hcy‐thiolactone, N‐Hcy‐protein, and Hcy on miR expression and on PHF8, H4K20me1, mTOR‐, and autophagy‐related proteins/mRNAs in HUVEC. Taken together, these findings show that Hcy metabolites upregulate miR‐22‐3p and miR‐1229‐3p expression, which then dysregulate the PHF8/H4K20me1/mTOR/autophagy pathway, important for vascular homeostasis. [ABSTRACT FROM AUTHOR]

Genetic or nutritional disorders in homocysteine (Hcy) or folate metabolism elevate plasma Hcy-thiolactone and lead to vascular and/or brain pathologies. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates N-Hcy-protein with autoimmunogenic and prothrombotic properties. Paraoxonase (PON1), carried on high-density lipoproteins (HDLs) in the blood, hydrolyzes Hcy-thiolactone and protects against the accumulation of N-Hcy-protein in vitro. To determine its role in vivo, we studied how natural variation in Hcy-thiolactonase activity of PON1 affects plasma NHcy- protein levels in cystathionine β-synthase-deficient patients (n=28). We found that plasma N-Hcy-protein was negatively correlated with serum Hcy-thiolactonase activity (r=-0.43, P=0.01), i.e., the higher the Hcythiolactonase activity, the lower N-Hcy protein levels. This relation was faithfully replicated in vitro in experiments with radiolabeled Hcy-thiolactone. We also found that enzymatic activities of the PON1 protein measured with artificial substrates correlated less strongly (r=-0.36, P=0.025 for paraoxonase activity) or did not correlate at all (phenylacetate hydrolase and TBLase activities) with plasma N-Hcy protein. These findings provide evidence that the Hcy-thiolactonase activity of PON1 is a determinant of plasma N-Hcyprotein levels and that Hcy-thiolactonase/PON1 protects proteins against N-homocysteinylation in vivo, a novel mechanism likely to contribute to atheroprotective roles of HDL in humans. [ABSTRACT FROM AUTHOR]

Genetic disorders of homocysteine (Hcy) or folate metabolism or high-methionine diets elevate plasma Hcy and its atherogenic metabolite Hcy-thiolactone. In humans, severe hyperhomocysteinemia due to genetic alterations in cystathionine β-synthase (Cbs) or methylenetetrahydrofolate reductase (Mthfr) results in neurological abnormalities and premature death from vascular complications. In mouse models, dietary or genetic hyperhomocysteinemia results in liver or brain pathological changes and accelerates atherosclerosis. Hcy-thiolactone has the ability to form isopeptide bonds with protein lysine residues, which generates modified proteins (N-Hcy-protein) with autoimmunogenic and prothrombotic properties. Our aim was to determine how N-Hcy-protein levels are affected by genetic or nutritional disorders in Hcy or folate metabolism in mice. We found that plasma N-Hcy-protein was elevated 10-fold in mice fed a high-methionine diet compared with the animals fed a normal commercial diet. We also found that inactivation of Cbs, Mthfr, or the proton-coupled folate transporter (Pcft) gene resulted in a 10- to 30-fold increase in plasma or serum N-Hcy-protein levels. Liver N-Hcy-protein was elevated 3.4-fold in severely and 11-fold in extremely hyperhomocysteinemic Cbs-deficient mice, 3.6-fold in severely hyperhomocysteinemic Pcft mice, but was not elevated in mildly hyperhomocysteinemic Mthfr-deficient animals, suggesting that mice have a capacity to prevent accumulation of N-Hcy-protein in their organs. These findings provide evidence that N-Hcy-protein is an important metabolite associated with Hcy pathophysiology in the mouse. [ABSTRACT FROM AUTHOR]

Severely elevated plasma homocysteine (Hcy) levels observed in genetic disorders of Hcy metabolism are associated with pathologies in multiple organs and lead to premature death due to vascular complications. In addition to elevating plasma Hcy, mutations in cystathionine β-synthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) gene lead to markedly elevated levels of circulating Hcy-thiolactone. The thiooester chemistry of Hcy-thiolactone underlies its ability to form isopeptide bonds with protein lysine residues (N-Hcy-protein), which may impair or alter the protein's function. However, it was not known whether genetic deficiencies in Hcy metabolism affect N-Hcyprotein levels in humans. Here we show that plasma N-Hcy-protein levels are significantly elevated in CBS and MTHFR-deficient patients. We also show that CBS deficient patients have significantly elevated plasma levels of prothrombotic Hcy-fibrinogen. These results provide a possible explanation for increased atherothrombosis observed in CBS-deficient patients. [ABSTRACT FROM AUTHOR]

Genetic disorders of homocysteine (Hcy) metabolism or a high-methionine diet lead to elevations of plasma Hcy levels. In humans, severe genetic hyperhomocysteinemia results in premature death from vascular complications whereas dietary hyperhomocysteinemia is often used to induce atherosclerosis in animal models. Hcy is mistakenly selected in place of methionine by methionyl-tRNA synthetase during protein biosynthesis, which results in the formation of Hcy-thiolactone and initiates a pathophysiological pathway that has been implicated in human vascular disease. However, whether genetic deficiencies in Hcy metabolism or a high-methionine diet affect Hcy-thiolactone levels in mammals has been unknown. Here we show that plasma Hcy-thiolactone is elevated 59-fold and 72-fold in human patients with hyperhomocysteinemia secondary to mutations in methylenetetrahydrofolate reductase and cystathionine β-synthase genes, respectively. We also show that mice, like humans, eliminate Hcy-thiolactone by urinary excretion; in contrast to humans, however, mice also eliminate significant amounts of plasma total Hey (∼38%) by urinary excretion. In mice, hyperhomocysteinemia secondary to a high-methionine diet leads to 3.7-fold and 25-fold increases in plasma and urinary Hcy-thiolactone levels, respectively. Thus, we conclude that hyperhomocysteinemia leads to significant increases in the atherogenic metabolite Hcy-thiolactone in humans and mice. [ABSTRACT FROM AUTHOR]