Melanie Brux

NADPH oxidase 4 protects against development of endothelial dysfunction and atherosclerosis in LDL receptor deficient mice

Genetic deletion of the hydrogen peroxide producing NADPH oxidase 4 (Nox4), as shown in the present study, leads to endothelial dysfunction and increased atherosclerosis under pathological conditions. Consequently, endothelial activation of Nox4 may represent a promising novel strategy for preventing endothelial dysfunction and atherosclerosis and its severe clinical complications. This also suggests that in contrast to the deleterious effects of oxidative stress certain reactive oxygen species might mediate beneficial effects in the vessel wall.

Lipoprotein apheresis of hypercholesterolemic patients mediates vasoprotective gene expression in human endothelial cells

OBJECTIVE Hypercholesterolemia is an important risk factor of cardiovascular diseases. Lipoprotein apheresis is an efficient strategy to reduce the serum low-density lipoprotein (LDL)-cholesterol and lipoprotein(a) levels and cardiovascular complications in patients with severe hypercholesterolemia. The underlying molecular mechanisms are not well-understood. In this study, we analyzed the impact of lipoprotein apheresis on gene expression in human endothelial cells. METHODS Human endothelial cells were stimulated with serum of hypercholesterolemic patients before and after lipoprotein apheresis. The expression of endothelial lipoprotein receptors, nitric oxide (NO) synthase and adhesion molecules was quantified by real-time PCR and Western blot. RESULTS Lipoprotein apheresis reduced the expression of the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) in endothelial cells. Low-density lipoprotein (LDL) receptor expression remained unchanged. The mRNA expression of the endothelial nitric oxide synthase (eNOS) was increased with serum of hypercholesterolemic patients after lipoprotein apheresis. In contrast, endothelial expression of vascular cell adhesion molecule 1 (VCAM-1) was reduced in response to serum after lipoprotein apheresis. CONCLUSION Lipoprotein apheresis reduced the expression of the proatherosclerotic oxLDL receptor LOX-1 and adhesion molecule VCAM-1 and increased the expression of vasoprotective and NO generating eNOS in human endothelial cells in response to serum of hypercholesterolemic patients. These novel molecular mechanisms may account for the antiatherosclerotic and vasoprotective potential of lipoprotein apheresis in patients with hypercholesterolemia.

Impact of Hey2 and COUP-TFII on genes involved in arteriovenous differentiation in primary human arterial and venous endothelial cells

Arteries and veins show marked differences in their anatomy, physiology and genetic expression pattern. In this study, we analyzed impact of overexpression or downregulation of arterial marker gene Hey2 and venous marker gene COUP-TFII in human venous and arterial endothelial cells on genes involved in arteriovenous differentiation. Lentiviral overexpression of venous marker gene COUP-TFII in arterial endothelial cells led to downregulation of NICD4, arterial marker gene Hey2 and EphrinB2. Downregulation of Hey2 could be mediated by direct binding of COUP-TFII to Hey2 promoter as shown by ChIP, EMSA and promoter analysis. Downregulation of Hey2 by shRNA causes downregulation of EphrinB2 expression. Overexpression of arterial marker Hey2 in venous endothelial cells did not change expression pattern of COUP-TFII. Downregulation of venous marker gene COUP-TFII in venous endothelial cells resulted in upregulation of VEGF-A, Dll4 and EphrinB2 expression. Our data support an important role of Hey2 and COUP-TFII in arteriovenous differentiation of human endothelial cells.

COUP-TFII is regulated by high glucose in endothelial cells.

Diabetes mellitus is an important risk factor for cardiovascular diseases. Clinical evidence supports a link between hyperglycemia, endothelial dysfunction, and vascular disorders. However, the precise molecular mechanisms causing endothelial dysfunction in diabetic patients remain unclear. An interesting novel mediator could be chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII), which plays an essential role in glucose metabolism. COUP-TFII is known to be expressed in venous endothelial cells. In this study, we show COUP-TFII expression in human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells. HUVECs express glucose transporters 1, 3, 6, and 10, and the insulin receptor. Insulin in combination with glucose activates protein kinase B (PKB or Akt) phosphorylation via phosphoinositide 3-kinase (PI3-kinase). Short-term (60-240 min) stimulation of HUVECs with high glucose increased COUP-TFII expression independent of insulin. Long-term (48 h) stimulation of HUVECs with high glucose augmented expression of the insulin receptor and E-selectin, but downregulated COUP-TFII protein expression. Downregulation of COUP-TFII by shRNA leads to downregulation of E-selectin and upregulation of eNOS and glucose transporters. Our data suggest that COUP-TFII is regulated by glucose in a time- and dose-dependent manner in endothelial cells. COUP-TFII might affect endothelial function in a diabetic background.

Cigarette smoke extract counteracts atheroprotective effects of high laminar flow on endothelial function

Tobacco smoking and hemodynamic forces are key stimuli in the development of endothelial dysfunction and atherosclerosis. High laminar flow has an atheroprotective effect on the endothelium and leads to a reduced response of endothelial cells to cardiovascular risk factors compared to regions with disturbed or low laminar flow. We hypothesize that the atheroprotective effect of high laminar flow could delay the development of endothelial dysfunction caused by cigarette smoking. Primary human endothelial cells were stimulated with increasing dosages of aqueous cigarette smoke extract (CSEaq). CSEaq reduced cell viability in a dose-dependent manner. The main mediator of cellular adaption to oxidative stress, nuclear factor erythroid 2-related factor 2 (NRF2) and its target genes heme oxygenase (decycling) 1 (HMOX1) or NAD(P)H quinone dehydrogenase 1 (NQO1) were strongly increased by CSEaq in a dose-dependent manner. High laminar flow induced elongation of endothelial cells in the direction of flow, activated the AKT/eNOS pathway, increased eNOS expression, phosphorylation and NO release. These increases were inhibited by CSEaq. Pro-inflammatory adhesion molecules intercellular adhesion molecule-1 (ICAM1), vascular cell adhesion molecule-1 (VCAM1), selectin E (SELE) and chemokine (C-C motif) ligand 2 (CCL2/MCP-1) were increased by CSEaq. Low laminar flow induced VCAM1 and SELE compared to high laminar flow. High laminar flow improved endothelial wound healing. This protective effect was inhibited by CSEaq in a dose-dependent manner through the AKT/eNOS pathway. Low as well as high laminar flow decreased adhesion of monocytes to endothelial cells. Whereas, monocyte adhesion was increased by CSEaq under low laminar flow, this was not evident under high laminar flow. This study shows the activation of major atherosclerotic key parameters by CSEaq. Within this process, high laminar flow is likely to reduce the harmful effects of CSEaq to a certain degree. The identified molecular mechanisms might be useful for development of alternative therapy concepts.