Objective: 

ARNO (Cytohesin-2) is a guanine nucleotide exchange factor important for cellular signalling through ADP-ribosylation factor GTPases. In endothelial cells vascular endothelial growth factor (VEGF) is an important stimulus of angiogenesis initiation. We investigated the influence of ARNO on VEGF dependent proliferation and signalling in endothelial cells as well as on VEGF dependent vessel permeability in vivo.

Methods: 

ARNO expression was detected by RT-PCR, Western Blot and immunohistochemical staining. ARNO inhibition in human microvascular endothelial cells (HMEC) was achieved by magnetofection of siRNA (30nM) or addition of the pharmacological cytohesin inhibitor SecinH3 (15mM). Protein activation was assessed by western blotting and proliferation was measured by MTT reduction. Vessel permeability was assessed in mouse cremaster muscle arterioles in vivo by extravasation of FITC-Dextran. VEGFR-2 surface expression was measured by flow cytometry.

Results: 

ARNO expression was detected in primary HUVEC and HMEC. Immunohistochemical staining of mouse aortae ex vivo and cell compartment fractioning showed a continous ARNO expression primarily on the luminal side and in the cellular membrane respectively (both n=3). VEGF (50ng/ml) dependent activation of Akt (p<0.05, n=6, HMEC) as well as proliferation (p<0.05, n=12, HMEC) was impaired after cytohesin inhibition (SecinH3) or ARNO siRNA treatment (p<0.05, n=3, HMEC and p<0.001, n=24, HMEC respectively). Moreover, ARNO inhibition reduced VEGF (10mg/ml) dependent vessel permeability in mouse cremaster muscle arterioles in vivo (p<0.05; n=4). Interestingly, ARNO siRNA treatment resulted in accelerated ligand induced (50ng/ml VEGF) reduction of VEGFR-2 cell surface expression (p<0.05, n=6, HMEC). Finally, ARNO siRNA treatment also decreased total VEGFR-2 expression levels (n=5, HMEC).

Conclusion: 

Our findings indicate a novel role for endothelial ARNO in VEGF dependent angiogenesis initiation by regulation of VEGFR-2 expression resulting in Akt pathway activation, vessel permeability and ultimately endothelial proliferation. Thus, ARNO may represent a new target for treatment of vascular disorders.