Regulation of NADPH oxidase 4 by hypoxia and flow in endothelial cells

Cardiovascular diseases are the leading cause of death worldwide. Risk factors like diabetes mellitus, hypertension and dyslipidemia are especially prevalent in developed countries. A modified cellular redox status has been demonstrated for all these risk factors. Oxidative stress in tissues is characterized by the increased formation of reactive oxygen species (ROS). They are produced by a variety of enzymes. In the vascular system, the group of NADPH Oxidases (NOX) is the most important source of free oxygen radicals. While most NADPH Oxidases produce superoxide (O₂˙^-), NOX4 is able to directly generate hydrogen-peroxide (H₂O₂), which is atheroprotective and acts as a direct vasodilator and second messenger in the cardiovascular system.

NOX4 is the highest expressed NADPH Oxidase in the vascular wall and its importance for vascular function is discussed controversially. In the context of cardiovascular diseases and tumorgenesis, a reduction in oxygen availability occurs. Hypoxia leads to changes in the expression profiles of specific marker genes. An important factor and indicator of hypoxia is the hypoxia-inducible factor (HIF). Furthermore, the induction of the vascular endothelial growth factor (VEGF) is a known downstream effect of hypoxia. The protective effect of NOX4 in the endothelium could play an important role in the improvement of the metabolic situation in the affected tissue in the context of hypoxia. A targeted regulation of NOX4 could therefore be a new therapeutic approach in the treatment of ischemic heart and tumor diseases.

The main aim of this work was to investigate the impact of hypoxia and atheroprotective flow on the expression of NOX4. Moreover, a possible regulation by mRNA stabilization or interaction with HIF1alpha and HIF2alpha was studied. For the experiments, endothelial cells of human umbilical veins (HUVEC) were used. Cultures at 21% O₂ were used as normoxic control samples. Following the experiments, RNA and protein were isolated and the expression of selected genes was evaluated using qPCR and Western Blot.

To analyze the effect of hypoxia on the mRNA expression of NOX 4, cells were incubated over a period of 96 h at an oxygen saturation of 1% O₂. It could be shown that hypoxia leads to a significant increase of NOX4 after 16 h. The expression of VEGF was also significantly increased and both HIF1alpha and HIF2alpha could be detected over a period of 2, 4, 8 and 24 h using Western Blot. HUVECs were incubated over a period of 16 and 24 h under 1%, 5% and 21% O₂. After 16 h under 1% O₂, a significant increase in mRNA expression of NOX4 and VEGF could be shown, while expression remained constant at 5% and 21% O₂. The protein expression of HIF1alpha and HIF2 alpha was also stronger detectable after 2, 4, 8 and 24 h using 1% O₂. Yet, under 5% and 21% O₂ there was no increased signal. Thus, hypoxia (1% O₂) leads to an induction of NOX4 on the mRNA level compared to expression under normoxic conditions (5% and 21% O₂). The cytostatic Actinomycin D was used to analyze a possible regulation of NOX4 expression by modifying mRNA stability. After 16 h of preincubation under normoxic and hypoxic conditions, Actinomycin D was applied at a concentration of 0,5 µg/ml and the cultures were further incubated for 4, 8 and 12 h at 21% and 1% O₂. Under hypoxia a slight increase in mRNA stability of the VEGF could be shown, but no change in mRNA stability of NADPH-Oxidase 4 was detectable. A regulatory mechanism depending on HIF1alpha and HIF2alpha was investigated by application of 1 mmol/l Dimethyloxalylglycin (DMOG) and incubation for 24 h at 21% O₂. Increased protein expression of HIF1alpha and HIF2alpha and a significant induction of mRNA expression of NOX4 and VEGF were shown. Hence, the regulation of NOX4 under hypoxia seems to be at least partly dependent on HIF1alpha and HIF2alpha.

In the second part of this thesis, the influence of atheroprotective flow under normoxic and hypoxic conditions on NOX4 expression was analyzed. Using the ibidi pump system, laminar, atheroprotective flow of 30 dyn/cm2 was applied and the cells incubated for 24 and 48 h at 21% and 1% O₂. The additional influence of atheroprotective flow led to a significant reduction of NOX4 mRNA expression under both normoxic and hypoxic conditions. In contrast, the mRNA expression of the endothelial NO synthase (eNOS) and VEGF was significantly increased. When hypoxia occurs under static conditions, the endothelium reacts with an induction of NOX4. The downstream effects of increased NOX4 mRNA expression, including the induction of angiogenesis by VEGF and the effect of H₂O₂ as a direct vasodilator and second messenger may improve the circulation and the metabolic status of the affected tissues. The regulation of NOX4 under hypoxia in HUVECs seems to be at least partially HIF-dependent.

A clear conclusion regarding regulation by increased mRNA stability could not be made. If hypoxia occurs in the presence of atheroprotective flow, the endothelial cells appear to respond primarily by an increase in the eNOS-NO signaling pathway and an upregulation of VEGF. On the other hand, the NOX4 expression is reduced. The interaction between HIF, VEGF, NOX4 and various NO synthases form a complex regulatory mechanism of endothelial function and the release of vasoactive substances. A better understanding of the molecular regulation and its interaction with other regulatory mechanisms could establish NOX4 as a novel therapeutic target.