Paracrine stimulation of endothelial cell motility and angiogenesis by platelet-derived deoxyribose-1-phosphate

Abstract

Objective: Micromolar concentrations of the proangiogenic metabolite deoxyribose-1-phosphate (dRP) were detected in platelet supernatants by mass spectrometry. In this study, we assessed whether the release of dRP by platelets stimulates endothelial cell migration and angiogenesis.

Methods and results: Protein-free supernatants from thrombin-stimulated platelets increased human umbilical vein endothelial cell migratory activity in transmigration and monolayer repair assays. This phenomenon was ablated by genetic silencing of dRP-generating uridine phosphorylase (UP) and thymidine phosphorylase (TP) or pharmacological inhibition of UP and restored by exogenous dRP. The stimulation of endothelial cell migration by platelet-derived dRP correlated with upregulation of integrin β(3), which was induced in a reactive oxygen species-dependent manner, and was mediated by the activity of the integrin heterodimer α(v)β(3). The physiological relevance of dRP release by platelets was confirmed in a chick chorioallantoic membrane assay, where the presence of this metabolite in platelet supernatants strongly induced capillary formation.

Conclusions: Platelet-derived dRP stimulates endothelial cell migration by upregulating integrin β(3) in a reactive oxygen species-dependent manner. As demonstrated by our in vivo experiments, this novel paracrine regulatory pathway is likely to play an important role in the stimulation of angiogenesis by platelets.

Figure 1.

Detection of dRP in platelet supernatants and whole cell extracts by mass spectrometry. (A) Gas chromatography-mass spectrometry single ion chromatogram (m/z 204) showing platelet extracts and a commercially available dRP standard. The dRP content of human platelet supernatants (B) or whole cell extracts (C) was investigated further by DIMS (from 4ml of platelet suspension). Thrombin-stimulated platelets were compared to resting platelets or plasma. The statistical significance of the differences was tested by one-way ANOVA or t-test (*= p<0.05, n=5).

Figure 2.

Release of dRP by mouse and human platelets stimulates HUVEC transmigration. (A) Schematic diagram of a transmigration chamber. (B) Complete (black bars) and protein-free supernatants (2 kDa cut-off; white bars) from resting and thrombin-stimulated platelets were added to the bottom of the transmigration chamber, and HUVEC transmigration was measured after 5 hrs. (C) Mouse platelets from WT (WT, white bars) and transgenic TP^−/−/UP^−/− platelets (grey bars) were treated with either vehicle solution (Tyrode’s buffer) or 1unit/ml thrombin for 5 minutes., whereas (D) human platelets were incubated with 100µM AEAC, 50µM PTAU, 50µM AG, or all three inhibitors (ALL) for 30 minutes, then treated with either vehicle solution or 0.1unit/ml thrombin for 5 minutes. Protein-free supernatants obtained as described were utilised for HUVEC transmigration assays (white bars). Where indicated 200µM dRP (grey bars) or 100µM AEAC, 50µM PTAU and 50µM AG (ALL, black bars) was added to protein-free supernatants immediately before use in the transmigration chambers. Transmigration was quantified after 5 hours and results were analysed by one-way ANOVA with Bonferroni post-test (*= p<0.05, n=4).

Figure 3.

Release of dRP by mouse platelets stimulates HUVEC monolayer repair in vitro. Platelets from WT and transgenic TP^−/−/UP^−/− mice were stimulated with 1unit/ml thrombin for 5 minutes. Vehicle solution (Tyrode’s buffer) or protein-free platelet supernatants were added to HUVECs after manual creation of a wound in the monolayer. Where indicated, 200µM dRP was added to the protein-free supernatants. Wound size at 0, 12, and 24 hours post wounding is expressed as surface area (pixels). Results from 4 independent experiments were analysed by two-way ANOVA with Bonferroni post-test (*= p<0.05, compared to WT supernatants).

Figure 4.

Platelet secretion of dRP induces integrin β3 upregulation in HUVECs in a ROS-dependent manner. HUVECs (A, B, F, and G) or HMVECs (C and D) were cultured for 6 hours in the presence of vehicle solution or protein-free supernatants from mouse platelets from WT or TP^−/−/UP^−/− animals (A, C, F, and G) or from human platelets treated with vehicle solution (resting) or stimulated with 1 unit/ml thrombin in the absence and presence of 50µM PTAU (B and D). Where indicated, 200µM dRP (A, B, D, and F) or 10mM N-acetyl-cysteine (G) were added to the supernatants immediately before incubation with HUVEC. After cell lysis, the expression of integrin β3 (A, B, C, D, F, and G) or HO-1 (F and G) were assessed by immunoblotting. All blots were also reprobed for actin. (E) The rate of intracellular ROS generation in HUVEC cells was measured using dihydroethidium (DHE, 10µM). Traces shown in panel i and ii indicate the rates of ROS production before and after addition of 100µM dRP or 1mM glucose, respectively. The rate of ROS generation in the absence or presence of dRP and glucose is measured as fluorescence unit/second and is presented as mean ± SEM (n=6) in panel iii. Statistical significance was tested by one-way ANOVA with Bonferroni post-test (*= p<0.05).

Figure 5.

Inhibition of integrin α_vβ₃ abolishes the stimulation of HUVEC monolayer repair by protein-free WT platelet supernatants. Protein-free supernatants were obtained from WT and TP^−/−/UP^−/− mouse platelets stimulated with 1unit/ml thrombin for 5 minutes. The supernatants were added to HUVECs after manual creation of a wound in the monolayer with and without supplementation with 12.5µg/ml LM609. Wound size at 0, 12, and 24 hours after wounding is expressed as surface area (pixels). The closure rate in the presence of vehicle (Tyrode’s buffer) is also represented by the dashed line. Results from 4 independent experiments were analysed by two-way ANOVA with Bonferroni post-test (*= p<0.05, compared to WT supernatants).

Figure 6.

Platelet-derived dRP stimulates angiogenesis in a chick chorioallantoic membrane (CAM) assay. Sterile filters soaked with vehicle, VEGF (100ng/filter) or the appropriate filtered platelet supernatant (2kDa cut off, 10⁹ platelets/ml) were applied to relatively avascular regions of the CAM and incubated for 48 hours before analysis of capillary formation as surface area occupied by the vascular network (angiogenic index). Representative images (A) and mean ± SEM from 15 CAMs/treatment (B) are shown (ANOVA with Bonferroni post-test, *= p<0.05).