Carom, a novel gene, is up-regulated by homocysteine through DNA hypomethylation to inhibit endothelial cell migration and angiogenesis

Abstract

Hyperhomocysteinemia (HHcy) is an independent risk factor for cardiovascular disease (CVD). We previously demonstrated that homocysteine (Hcy) suppresses endothelial cell (EC) proliferation, migration, and post-injury EC repair, but the molecular mechanism underlying Hcy-induced EC injury is unclear. In this study, we identified a novel gene, Carom, which mediates Hcy-induced suppression of EC migration and angiogenesis. We identified FCH and double SH3 domains 2 (FCHSD2), a novel gene, as an Hcy-responsive gene through Differential Display in Hcy (50µM)-treated human umbilical vein endothelial cells (HUVEC). FCHSD2 was initially named as Carom, based on the identification of this molecule as an interacting protein of calcium/calmodulin-dependent serine protein kinase (CASK) and membrane associated guanylate kinase, WW and PDZ domain containing 1 (MAGI1). In this thesis, we describe this gene as Carom. Carom belongs to the Fes/CIP4 homology and Bin/amphiphysin/Rvs (F-BAR) protein family, which is a group of multivalent adaptors linking plasma membrane and cytoskeleton, involved in endocytosis and cell migration. However, Carom's function is poorly characterized. Based on the findings that CASK and MAGI1 inhibit cell migration and growth, and the role of F-BAR proteins in cell migration, we hypothesize that Hcy up-regulates Carom to inhibit EC growth and/or migration, finally leading to CVD. We confirmed the significant induction of Carom mRNA expression in Hcy-treated HUVECs or human aortic endothelial cells (HAEC) by Northern blot and Real-time PCR. In addition, we found that Carom protein expressions were significantly increased both in Hcy-treated HAECs and lung ECs isolated from HHcy mice by Western blot using our homemade rabbit antibody against Carom. These data indicate that Hcy increases endothelial expression of Carom both in vitro and in vivo. Furthermore, in order to characterize Carom function in EC, we generated recombinant adenovirus Adv-Carom to transduce Carom for gain-of-function study and Adv-Carom-shRNA to express Carom shRNA for loss-of-function study. We found that neither adenovirus-transduced Carom expression nor adenoviral Carom shRNA had any impact on HUVEC proliferation by using [3H]-thymidine incorporation. Interestingly, we demonstrated that Adv-Carom inhibited HAEC migration, while Hcy-induced HEAC migration inhibition could be rescued by Adv-Carom-shRNA. These data suggest that Carom may inhibit angiogenesis via a cell proliferation-independent mechanism. Furthermore, we found that Hcy significantly increased the intracellular level of S-adenosyl homocysteine (SAH) but not S-adenosyl methionine (SAM), and decreased the SAM/SAH ratio, an indicator of cellular methylation, in HAECs, by using High-performance liquid chromatography/electrospray tandem mass spectrometry (HPLC-MS) to measure SAH and SAM levels. Meanwhile, Carom protein expression was significantly induced by azacytidine (AZC), a DNA methyltransferse inhibitor, in a dose-dependent manner in HAECs. Based on these data, we speculated that Hcy-induced hypomethylation could associate with Carom up-regulation. Thus we used bisulfite deep sequencing to profile methylation status of Carom gene in Hcy-treated HUVECs and found that Carom promoter was hypomethylated by Hcy. In addition, eight transcriptional factor binding sites on Carom were hypomethylated by Hcy. These data suggest that Hcy may induce Carom via a DNA hypomethylation-dependent mechanism. Moreover, we found that adenovirus-transduced Carom expression significantly increased the secretions of two anti-angiogenic chemokines, CXCL10 and CXCL11 in HAECs by using human cytokine array. Similarly, Hcy also significantly increased mRNA expressions of CXCL10 and CXCL11, while Adv-Carom-shRNA blocked down the inductions of CXCL10 and CXCL11 by Hcy. We further demonstrated that adenovirus-transduced Carom expression inhibited angiogenesis by performing tube formation assay of HAECs, whereas Hcy-induced angiogenesis suppression were rescued by Adv-Carom-shRNA as well as the neutralizing antibodies of CXCL10 and CXCL11. These data suggest that Hcy induces Carom to trigger CXCL10 and CXCL11 downstream to inhibit angiogenesis. In conclusion, Hcy induces Carom expression through DNA hypomethylation to inhibit EC migration and angiogenesis.