What can transcriptomics tell us about cell responses to elevated homocysteine?

Epidemiological studies have shown that elevated plasma homocysteine increases the risk of cardiovascular diseases, such as myocardial infarction, stroke and coronary artery dis-ease. At the molecular level, high homocysteine promotes the generation of reactive oxygen species, decreases nitric oxide bioavailability, alters lipid metabolism and initiates inflamma-tory responses. These alterations cause physiological dysfunction of the endothelium, which leads to atherosclerosis. Deposition of lipids within the arterial wall is a major characteristic of atherosclerosis, as such we hypothesized that elevated homocysteine affects the regula-tion of hepatic lipid metabolism. However, the molecular mechanisms that mediate the dys-function of the endothelium are not well defined. Advances in sequencing technology now allow us to perform high resolution profiling of gene expression using a technique called RNA-seq. Here we apply RNA-seq to understand hepatocyte (Hep G2) and endothelial cell (HMEC-1) responses to 200 μM homocysteine for 3 days. We observed increased strength of responses in Hep G2 cells which can be attributed to its higher metabolic nature. Our data demonstrates that homocysteine alters cholesterol biosynthesis in Hep G2 cells through the activation of SREBP, which other studies have found to be caused by ER stress. Homocysteine up-regulates haemostasis and down-regulates the formation of fibrin clot in Hep G2 cells. Homocysteine up-regulates the IL-6 receptor and components of JAK/STAT sig-nalling pathway in Hep G2 cells. Homocysteine enhances the cytotoxic damage potential of ROS by down-regulating the expression of antioxidant enzymes thioredoxin, glutathione pe-roxidase, superoxide dismutase, peroxiredoxin and catalase. Multiple pathways of NF-κB ac-tivation and signalling were down-regulated through the down-regulation of genes encoding ubiquitin and proteasomal subunits. In summary, we have identified novel alterations in the cellular responses due to elevated homocysteine, which provide more insight into the mo-lecular mechanisms that mediate homocysteine-induced endothelial dysfunction. This work will enable future studies into these novel mechanisms, such as investigations into the al-tered haemostasis or lipid profiles. Furthermore, these studies will allow us to better under-stand the pathogenesis of elevated homocysteine.