[Frontiers in Bioscience E3, 955-967, June 1, 2011]

Hypoxia-induced ROS signaling is required for LOX up-regulation in endothelial cells

Anna Guadall1, Mar Orriols1, Javier F. Alcudia1, Victoria Cachofeiro2, Jose Martinez-Gonzalez1, Cristina Rodriguez1

1Centro de Investigacion Cardiovascular (CSIC-ICCC), Hospital de la Santa Creu i Sant Pau, c/ Antoni MĒ Claret 167, 08025 Barcelona, Spain. 2Departamento de Fisiologia, Facultad de Medicina, Universidad Complutense, Avda. de la Complutense s/n, 28040 Madrid, Spain

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture
3.2. Real-time PCR
3.3. Immunostaining and confocal microscopy
3.4. Lysyl oxidase activity
3.5. Constructs of LOX promoter
3.6. Transient transfections and luciferase assays
3.7. HIF-1alpha siRNA transfection
3.8. Western blot analysis
3.9. Statistical analysis
4. Results
4.1. Hypoxia induces LOX expression in endothelial cells
4.2. The up-regulation of LOX by hypoxia is independent of autocrine factors
4.3. Hypoxia increases LOX transcriptional activity in endothelial cells
4.4. Rapamycin partially prevented the induction of LOX by hypoxia
4.5. HIF-1alpha inhibition partially abrogated hypoxia-induced LOX up-regulation
4.6. ROS signaling is involved in hypoxia-induced LOX up-regulation
4.7. Smad pathway is involved in the increase of LOX transcriptional activity induced by hypoxia
5. Discussion
6. Acknowledgment
7. References

1. ABSTRACT

The adaptive response of endothelial cells to hypoxia involves a substantial remodeling of extracellular matrix (ECM). In endothelial cells hypoxia up-regulates lysyl oxidase (LOX), a key enzyme in ECM assembly, relevant to vascular homeostasis. However, the mechanism underlying this response has not been established. Hypoxia up-regulated LOX expression in endothelial cells (HUVEC and BAEC) and concomitantly increased LOX enzymatic activity. This effect was independent of autocrine factors released by hypoxic cells and relies on a transcriptional mechanism. Both mTOR blockade and HIF-1alpha knockdown slightly prevented LOX up-regulation by hypoxia, suggesting that HIF-1alpha is only partially responsible for this effect. In fact, serial promoter deletion and mutagenesis studies indicated a limited contribution of the previously described hypoxia response element (-75 bp). Interestingly, Smad over-expression further increased LOX transcriptional activity in endothelial cells exposed to hypoxia. Moreover, the increase in LOX expression triggered by hypoxia was significantly reduced by reactive oxygen species (ROS) inhibitors. Thus, our data support a role of Smad signaling and ROS in the up-regulation of LOX by hypoxia in endothelial cells.