Homocysteine metabolites impair the PHF8/H4K20me1/mTOR/autophagy pathway by upregulating the expression of histone demethylase PHF8‐targeting microRNAs in human vascular endothelial cells and mice

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. Hypothetical pathway leading to autophagy inhibition and endothelial dysfunction in hyperhomocysteinemia. Hcy metabolites upregulate miR‐22‐3p and miR‐1229‐3p in human endothelial cells and miR22‐3p in Cbs−/− mouse heart. These miRs downregulate the histone demethylase PHF8, thereby elevating H4K20me1, a positive regulator of mTOR. Upregulation of mTOR and pmTOR inhibits autophagy in human endothelial cells and mouse heart. Up and down arrows show the direction of changes. Hcy, homocysteine; mTOR, mammalian target of rapamycin; pmTOR, phospho‐mTOR; PHF8, Plant Homeodomain Finger protein 8.