Platelets modulate gastric ulcer healing: role of endostatin and vascular endothelial growth factor release

Abstract

Bleeding and delayed healing of ulcers are well recognized clinical problems associated with the use of aspirin and other nonsteroidal antiinflammatory drugs, which have been attributed to their antiaggregatory effects on platelets. We hypothesized that antiplatelet drugs might interfere with gastric ulcer healing by suppressing the release of growth factors, such as vascular endothelial growth factor (VEGF), from platelets. Gastric ulcers were induced in rats by serosal application of acetic acid. Daily oral treatment with vehicle, aspirin, or ticlopidine (an ADP receptor antagonist) was started 3 days later and continued for 1 week. Ulcer induction resulted in a significant increase in serum levels of VEGF and a significant decrease in serum levels of endostatin (an antiangiogenic factor). Although both aspirin and ticlopidine markedly suppressed platelet aggregation, only ticlopidine impaired gastric ulcer healing and angiogenesis as well as reversing the ulcer-associated changes in serum levels of VEGF and endostatin. The effects of ticlopidine on ulcer healing and angiogenesis were mimicked by immunodepletion of circulating platelets, and ticlopidine did not influence ulcer healing when given to thrombocytopenic rats. Incubation of human umbilical vein endothelial cells with serum from ticlopidine-treated rats significantly reduced proliferation and increased apoptosis, effects reversed by an antibody directed against endostatin. Ticlopidine treatment resulted in increased platelet endostatin content and release. These results demonstrate a previously unrecognized contribution of platelets to the regulation of gastric ulcer healing. Such effects likely are mediated through the release from platelets of endostatin and possibly VEGF. As shown with ticlopidine, drugs that influence gastric ulcer healing may do so in part through altering the ability of platelets to release growth factors.

Figures

Figure 1

Effects of ticlopidine and aspirin on gastric ulcer healing (A) and angiogenesis in the granulation tissue (B) 10 days after ulcer induction. Three days after ulcer induction, the mean ulcer size was 78 ± 8 mm2. The rats were given vehicle, ticlopidine (100 or 300 mg/kg), or aspirin (30 mg/kg) orally each day from day 3 to day 9 postulcer induction. Ulcer healing is expressed as a percent reduction in ulcer size from that on day 3. *, P < 0.05; **, P < 0.01 vs. the corresponding vehicle-treated group (the statistical analysis was performed by using the raw ulcer area data).

Figure 2

Platelet aggregation induced by thrombin (A), arachidonic acid (B), and ADP (C). Rats were given vehicle, ticlopidine (300 mg/kg), or aspirin (30 mg/kg) orally each day for 7 days. Three hours after the final dose, platelet-rich plasma was prepared and platelet aggregation in response to the three agonists was assessed. *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding control group.

Figure 3

Effects of ticlopidine and aspirin on serum VEGF (A) and endostatin (B) levels. Beginning 3 days after ulcer induction, rats were given vehicle, ticlopidine (300 mg/kg), or aspirin (30 mg/kg) orally each day for 7 days. The “No ulcer” group consisted of sham-operated rats that were treated each day with vehicle. *, P < 0.05; **, P < 0.01 vs. the corresponding vehicle-treated group. #, P < 0.01 vs. the “No ulcer” group.

Figure 4

Effects of serum derived from ticlopidine- or aspirin-treated rats on HUVEC proliferation (A) and apoptosis (B). HUVECs were incubated with sera from vehicle-treated, ticlopidine-treated (300 mg/kg), or aspirin-treated (30 mg/kg) rats for 24 h. Cell proliferation was determined by the MTT assay, and apoptosis was assessed by DAPI staining (B). *, P < 0.05; **, P < 0.01; ***, P < 0.001 vs. the corresponding vehicle-treated group. The dotted lines show the mean levels of proliferation or apoptosis in cells incubated only with the control medium (containing 2.5% FBS, but no rat serum).

Figure 5

Effects of antiendostatin, anti-VEGF, and control antiserum (IgG) on HUVEC proliferation (A) and apoptosis (B; DAPI staining) after exposure to 5% rat serum. Serum from ticlopidine-treated rats reduced proliferation and increased apoptosis (***, P < 0.001). Antiendostatin abolished the effects of the serum from ticlopidine-treated rats (#, P < 0.05 vs. the “ticlopidine alone” and ticlopidine + IgG groups).

Figure 6

Release of VEGF (A) and endostatin (B) from platelets in response to stimulation with ADP or thrombin. Platelets were harvested from rats that had been treated daily for 1 week with vehicle, ticlopidine (300 mg/kg), or aspirin (30 mg/kg) and then were challenged in vitro with saline, ADP (10 μM), or thrombin (0.75 unit/ml). *, P < 0.05; **, P < 0.01 vs. corresponding vehicle-treated control; ##, P < 0.01 vs. the corresponding saline-challenged group.