Evidence that platelets promote tube formation by endothelial cells on matrigel

Abstract

1 The involvement of platelets in neovascularization was investigated in the matrigel tube formation assay, an in vitro model of angiogenesis. 2 Platelets promoted the formation of capillary-like structures (expressed as relative tube area) numberand time-dependently. Relative tube area increased from 0.98+0.02 (n=8) in the presence of 6.25610 4 , to 3.21+0.12 (n=8) in the presence of 10 6 platelets/well compared to 0.54+0.04 (n=8) in their absence. This increase was unaected by acetyl salicylic acid (ASA), apyrase, and hirudin. Photographs from representative experiments, showed that platelets adhered along the dierentiating endothelium. 3 Addition of a-thrombin (0.1 ± 1 i.u. ml 71 ), the nitric oxide (NO) donor sodium nitroprusside (SNP; 1 ± 100 mM) or the NO synthase inhibitor, L-NG-arginine-methylester (L-NAME, 30 ± 300 mM) to the assay, had no eect on tube formation compared to that seen with platelets alone. 4 Neuraminidase (0.01 i.u./10 7 platelets), which strips sialic acid residues from membrane glycoproteins, abolished the promoting eect of platelets on tube formation. The relative tube area in the presence of neuraminidase-treated platelets was 0.81+0.03 (n=8), in the presence of untreated platelets 1.69+0.09, P50.001 (n=8) and in the absence of platelets, 0.80+0.04 (n=8). The tetrapeptide Arg-Gly-Asp-Ser (RGDS; 20 ± 200 mM) which inhibits von Willebrand factor, ®brinogen and ®bronectin-mediated adhesion, had no eect on the promoting eect of platelets on tube formation. 5 These results indicate that platelets promote angiogenesis in vitro. This eect is largely independent from activation by a-thrombin, is not modi®ed by manipulating NO and prostaglandin metabolism and proceeds possibly through adhesion of the platelets to the dierentiating endothelium.

Activated Platelets Provide a Functional Microenvironment for the Antiangiogenic Fragment of Histidine-Rich Glycoprotein

“…Besides their role in hemostasis, platelets contribute to nonhemostatic processes such as immunity, tumor metastasis, and angiogenesis (9)(10)(11). Despite their content of both positive and negative regulators of blood vessel formation, platelets have in several different experimental settings been shown to stimulate angiogenesis (11)(12)(13)(14)(15). This apparent contradiction may be explained by recent data from Italiano and coworkers (16).…”

Heparin affin regulatory peptide binds to vascular endothelial growth factor (VEGF) and inhibits VEGF-induced angiogenesis

“…The solidified Matrigelt was then overlaid with 500 ml EBM-2 containing 8 Â 10 4 HUVEC supplemented with 2% FCS with or without growth factors. After incubation at 371C for 4-6 h, tube network formation was quantified as previously described (Pipili-Synetos et al, 1998).…”

Platelets and platelet adhesion support angiogenesis while preventing excessive hemorrhage

“…The potential role of platelets in angiogenesis was first suggested by the in vitro observations that platelets stimulated endothelial cell proliferation in culture and promoted formation of capillary-like structures in Matrigel assays (15). The early studies led to the identification of many pro-and antiangiogenic factors that are stored in platelets and released after platelet activation.…”