Adenosine stimulates canine retinal microvascular endothelial cell migration and tube formation

Purpose. To evaluate the effects of adenosine and related substances on events that occur during vasculogenesis and angiogenesis, using in vitro assays. Methods. Adenosine (ADO), inosine (INO, an adenosine catabolite), and 5'-(N-ethylcarboxamido) adenosine (NECA, an adenosine agonist) were evaluated for their effect on the proliferation of canine retinal microvascular endothelial cells (DRME), using a cell count assay. Also, these substances and ADO receptor selective agonists and antagonists were evaluated in an assay for DRME chemokinesis by measuring random migration into a wound made in a confluent cellular monolayer. Finally, the effects of these substances on DRME cord formation were evaluated in a 3-dimensional collagen gel. Bovine retinal extract (RE) was used as a positive control for all assays. Results. There was no effect on proliferation of DRME by any of the substances related to adenosine, but VEGF yielded a 30% stimulation of proliferation. Retinal extract, 10 μM ADO and 1.2 nM VEGF stimulation of DRME migration was 2- to 2.5-fold greater than 10 μM INO yielded. In addition, a combination of 1.2 nM VEGF with 10 μM ADO exceeded the stimulation in migration by ADO only and VEGF only. The total length of tubes formed in the presence of 10 μM ADO was comparable to that formed in the presence of RE and was 11-fold greater than with 10 μM INO. Tube length with a combination of VEGF plus ADO was 36% greater than with retinal extract. Use of selective ADO receptor antagonists suggested that tube formation and the migration response may be mediated through both adenosine A₁ and A₂ receptors, but use of selective ADO agonists suggests that A₂ receptors may be more important than A₁ for endothelial cell migration. Conclusions. This in vitro analysis suggests that adenosine may stimulate retinal vasculogenesis, an event which involves migration of angioblasts and their assembly into vascular cords, prior to canalization.