NOX5 IS INVOLVED IN ANGIOTENSIN-II-INDUCED ARTERIAL FRACTALKINE (CX3CL1) EXPRESSION AND MONONUCLEAR CELL ARREST

C Rius1,2, L Piqueras2, F Albertos1,2, H González2, P Escudero1,2, EJ Morcillo1,2, MJ Sanz1,2. 1University of Valencia, Pharmacology, Spain, 2University Clinic Hospital Research Foundation-INCLIVA, INCLIVA, Spain

Background: The production of Reactive Oxigen Species (ROS) and activation of redox-sensitive signalling pathways mediate many of the inflammatory responses induced by angiotensin-II (Ang-II). On the other hand, in previous studies we have demonstrated that Ang-II promotes arterial fractalkine (CX3CL1)-dependent mononuclear cell recruitment.

Objective: Potential sources of superoxide anion in the vasculature include the activation of NADPH oxidases (Nox) and xantine oxidase (XO). Since Nox2, Nox4, and Nox5 are the main NADPH oxidase isoforms in endothelial cells, we have investigated the enzymes involved in Ang-II induced CX3CL1 expression and the subsequent mononuclear cell recruitment.

Methods: Human umbilical arterial endothelial cells (HUAECs) were stimulated with Ang-II (1 μM, 24h) in the presence or absence of apocynin (NADPH oxidase inhibitor, 30 μM) or allopurinol (XO inhibitor, 100 μM). In some experiments, cells were transfected with a specific small interference RNA (siRNA) to knock-down Nox2, Nox4 and Nox5 expression or with its respective control using lipofectamine RNAiMAX. Flow cytometry was employed to determine endothelial CX3CL1 expression. Finally, flow chamber assay was used to determine mononuclear cell arrest under dynamic conditions. Isolated human mononuclear cells (1 x 106 cells/ml) were perfused over the endothelial monolayers for 5 min at 0.5 dyn/cm2, and mononuclear leukocyte recruitment quantified. Differences between two groups were determined by paired or unpaired Student’s t test, as appropriate. Data were considered statistically significant when p<0.05.

Results: Preincubation of endothelial cells with apocynine inhibited Ang-II-induced CX3CL1 protein expression and mononuclear cell adhesion by 93.6±4.7% (n=7, p<0.01) and 77.3±6.7% (n=7, p<0.01) respectively. In contrast, pretreatment of endothelial cells with the XO inhibitor allopurinol showed no effect on these parameters (n=7). Forty-eight hours posttransfection with Nox2, Nox4 or Nox5-specific siRNA, HUAECs showed a >70% reduction in mRNA and protein expression, compared with levels detected in the control siRNA-treated cells. Interestingly, while knockdown of Nox5 abrogated the increase in CX3CL1 expression and mononuclear cell recruitment caused by Ang-II (93.0±7.0 and 98.0±2.0% inhibition respectively, n=6, p<0.01) neither Nox2 nor Nox4 silencing affected these responses (n=6).

Conclusions: These results suggest that Nox5 is involved in Ang-II-induced CX3CL1 expression on arterial endothelial cells and the subsequent mononuclear cell recruitment. The design of new drugs targeting CX3CL1 or Nox5 function might reduce the vascular inflammation associated with rennin-angiotensin system activation.

This work was supported by grants SAF2009-08913, SAF2011-23777, CP07/00179, PI/08/1875 and RIER RD08/0075/0016, from Spanish Ministry of Science and Innovation, Carlos III Health Institute, Spanish Ministry of Health and several grants from Generalitat Valenciana.