Oxidative stress occurs with disturbed blood flow, inflammation and cardiovascular disease (CVD), yet free-radical scavenging antioxidants have shown limited benefit in human CVD. Thioredoxin-1 (Trx1) is a thiol antioxidant protecting against non-radical oxidants by controlling protein thiol/disulfide status; Trx1 translocates from cytoplasm to cell nuclei due to stress signaling, facilitates DNA binding of transcription factors, e.g., NF-κB, and potentiates inflammatory signaling. Whether increased nuclear Trx1 contributes to proatherogenic signaling is unknown.
Here, we investigated whether hyperglycemia and/or free fatty acids (palmitate, PAL) affect the expression level of bone morphogenic protein 4 (BMP4), a proatherogenic marker, in endothelial cells and the potential role of BMP4 in diabetic vascular complications. To measure BMP4 expression, human umbilical vein endothelial cells (HUVECs) were exposed to high glucose concentrations and/or PAL for 24 or 72 h, and the effects of these treatments on the expression levels of adhesion molecules and reactive oxygen species (ROS) were examined. BMP4 loss-of-function status was achieved via transfection of a BMP4-specific siRNA. High glucose levels increased BMP4 expression in HUVECs in a dose-dependent manner. PAL potentiated such expression. The levels of adhesion molecules and ROS production increased upon treatment with high glucose and/or PAL, but this effect was negated when BMP4 was knocked down via siRNA. Signaling of BMP4, a proinflammatory and pro-atherogenic cytokine marker, was increased by hyperglycemia and PAL. BMP4 induced the expression of inflammatory adhesion molecules and ROS production. Our work suggests that BMP4 plays a role in atherogenesis induced by high glucose levels and/or PAL.
Epigenetic mechanisms that regulate endothelial cell gene expression are now emerging. DNA methylation is the most stable epigenetic mark that confers persisting changes in gene expression. Not only is DNA methylation important in rendering cell identity by regulating cell type-specific gene expression throughout differentiation, but it is becoming clear that DNA methylation also plays a key role in maintaining endothelial cell homeostasis and in vascular disease development. Disturbed blood flow causes atherosclerosis, whereas stable flow protects against it by differentially regulating gene expression in endothelial cells. Recently, we and others have shown that flow-dependent gene expression and atherosclerosis development are regulated by mechanisms dependent on DNA methyltransferases (1 and 3A). Disturbed blood flow upregulates DNA methyltransferase expression both in vitro and in vivo, which leads to genome-wide DNA methylation alterations and global gene expression changes in a DNA methyltransferase-dependent manner. These studies revealed several mechanosensitive genes, such as HoxA5, Klf3, and Klf4, whose promoters were hypermethylated by disturbed blood flow, but rescued by DNA methyltransferases inhibitors such as 5Aza-2-deoxycytidine. These findings provide new insight into the mechanism by which flow controls epigenomic DNA methylation patterns, which in turn alters endothelial gene expression, regulates vascular biology, and modulates atherosclerosis development.
Atherosclerosis is the leading cause of morbidity and mortality in the U.S., and is a multifactorial disease that preferentially occurs in regions of the arterial tree exposed to disturbed blood flow. The detailed mechanisms by which d-flow induces atherosclerosis involve changes in the expression of genes, epigenetic patterns, and metabolites of multiple vascular cells, especially endothelial cells. This review presents an overview of endothelial mechanobiology and its relation to the pathogenesis of atherosclerosis with special reference to the anatomy of the artery and the underlying fluid mechanics, followed by a discussion of a variety of experimental models to study the role of fluid mechanics and atherosclerosis. Various in vitro and in vivo models to study the role of flow in endothelial biology and pathobiology are discussed in this review. Furthermore, strategies used for the global profiling of the genome, transcriptome, miR-nome, DNA methylome, and metabolome, as they are important to define the biological and pathophysiological mechanisms of atherosclerosis. These "omics" approaches, especially those which derive data based on a single animal model, provide unprecedented opportunities to not only better understand the pathophysiology of atherosclerosis development in a holistic and integrative manner, but also to identify novel molecular and diagnostic targets.
8-Hydroxy-2-deoxyguanosine (8-OHdG), a marker of oxidative stress, has been recently rediscovered to inhibit Rac1 in neutrophils and macrophages, thereby inhibiting Rac1-linked functions of these cells, including reactive oxygen species production through NADPH oxidase activation, phagocytosis, chemotaxis, and cytokine release. In vascular smooth muscle cells (VSMCs), reactive oxygen species also induce abnormal proliferation and migration leading to progression of atherosclerosis. Based upon the involvement of reactive oxygen species in phagocytic cells and VSMCs during the atherosclerotic process, we hypothesized that 8-OHdG could have antiatherosclerotic action and tested this hypothesis in an experimentally induced atherosclerosis in mice. Partially ligated ApoE knockout mice, a more physiologically relevant model of low and oscillatory flow, developed an advanced lesion in 2 weeks, and orally administered 8-OHdG significantly reduced plaque formation along with reduced superoxide formation, monocyte/macrophage infiltration, and extracellular matrix (ECM) accumulation. The effects of 8-OHdG observed in primary VSMCs were consistent with the in vivo effects of 8-OHdG and were inhibitory to angiotensin II or platelet-derived growth factor-induced production of reactive oxygen species, proliferation, migration, and ECM production. Also, angiotensin II-induced Rac1 activity in VSMCs was significantly inhibited by 8-OHdG, and transfection of constitutively active Rac1 reversed the inhibitory effect of 8-OHdG on VSMC activation. Molecular docking study showed that 8-OHdG stabilizes Rac1-GEF complex, indicating the physical contact of 8-OHdG with Rac1. These findings highly suggest that the antiatherosclerotic effect of 8-OHdG is mediated by inhibition of Rac1 activity. In conclusion, our results show a novel action of orally active 8-OHdG in suppressing atherosclerotic plaque formation in vivo and VSMC activation in vitro through inhibition of Rac1, which emphasizes a new therapeutic avenue to benefit atherosclerosis.
Objective-Inflammation plays a central role in atherosclerosis. However, the detailed changes in the composition and quantity of leukocytes in the arterial wall during atherogenesis are not fully understood in part because of the lack of suitable methods and animal models.
Methods and Results-We developed a 10-fluorochrome, 13-parameter flow cytometry method to quantitate 7 major leukocyte subsets in a single digested arterial wall sample. Apolipoprotein E-deficient mice underwent left carotid artery (LCA) partial ligation and were fed a high-fat diet for 4 to 28 days. Monocyte/macrophages, dendritic cells, granulocytes, natural killer cells, and CD4 T cells significantly infiltrated the LCA as early as 4 days. Monocyte/macrophages and dendritic cells decreased between 7 and 14 days, whereas T-cell numbers remained steady. Leukocyte numbers peaked at 7 days, preceding atheroma formation at 14 days. B cells entered LCA by 14 days. Control right carotid and sham-ligated LCAs showed no significant infiltrates. Polymerase chain reaction and ELISA arrays showed that expression of proinflammatory cytokines and chemokines peaked at 7 and 14 days postligation, respectively.
Conclusion-This is the first quantitative description of leukocyte number and composition over the life span of murine atherosclerosis. These results show that disturbed flow induces rapid and dynamic leukocyte accumulation in the arterial wall during the initiation and progression of atherosclerosis.
Atherosclerosis is a multifactorial disease that preferentially occurs in arterial regions exposed to d-flow can be used to indicate disturbed flow or disturbed blood flow. The mechanisms by which d-flow induces atherosclerosis involve changes in the transcriptome, methylome, proteome, and metabolome of multiple vascular cells, especially endothelial cells. Initially, we begin with the pathogenesis of atherosclerosis and the changes that occur at multiple levels owing to d-flow, especially in the endothelium. Also, there are a variety of strategies used for the global profiling of the genome, transcriptome, miRNA-ome, DNA methylome, and metabolome that are important to define the biological and pathophysiological mechanisms of endothelial dysfunction and atherosclerosis. Finally, systems biology can be used to integrate these ‘omics’ datasets, especially those that derive data based on a single animal model, in order to better understand the pathophysiology of atherosclerosis development in a holistic manner and how this integrative approach could be used to identify novel molecular diagnostics and therapeutic targets to prevent or treat atherosclerosis. WIREs Syst Biol Med 2016, 8:378–401. doi: 10.1002/wsbm.1344. For further resources related to this article, please visit the WIREs website.