Noval Insights into the Role of Platelet Angiogenic Growth Factors on the Regulation of Normal Vascular Development

The role of platelets as angiogenic regulators has been the subject of intense research over the last decade, and has led to the recognition of platelets as the predominant source of key angiogenic growth factors (AGFs) in the circulation. Through the regulated delivery of these factors, platelets contribute to wound healing, maintenance of vascular integrity, and tumor angiogenesis. Prior studies have shown that thrombocytopenia in adult mice leads to hemorrhages primarily from structurally abnormal new blood vessels, both in in vivo angiogenesis assays and inside tumors. The rapid growth during fetal and neonatal life is associated with a developmental stage-specific need for accelerated angiogenesis and vascular development. However, whether and how platelets participate in normal fetal/neonatal vascular development is unknown, except for their role in blood/lymphatic separation. We hypothesized that platelets would be important for normal fetal/neonatal vascular development, and that their AGF profile would be different from that of adult platelets. As a first step, we quantified five key AGFs (VEGF-A, bFGF, ANG-1, PDGF-BB and TGF-b) by ELISA in platelet lysates and platelet-poor plasma from term cord blood samples (CB; n=12) and from the peripheral blood of healthy adult volunteers (PB; n=8). These studies demonstrated that CB platelets, just like PB platelets, are the major source of AGFs in the circulation, carrying more than 80% of the levels of these AGFs present in the blood. However, the angiogenic profiles were different in platelets of different developmental origins. Fetal/neonatal platelets contained ~70% more bFGF and VEGF-A than adult platelets (p<0.01 for both), but had significantly lower levels of ANG-1, PDGF-BB and TGF-β1 (p<0.05 vs. adult platelets). VEGF-A and bFGF are essential for the proliferation and differentiation of endothelial cells, while ANG-1, PDGFs and TGF-b concert mural cell coverage, basement membrane formation and vascular maturation, thus stabilizing and "strengthening" the vasculature. Based on these characteristics, we hypothesized that neonatal and adult platelets would have different biological effects. To test this, we first assessed the effects of CB- vs. PB-derived platelet lysates on human umbilical vascular endothelial cells (HUVEC), in a tube formation assay. In these experiments, CB platelets more potently stimulated angiogenesis, quantified as number of capillary branch points at 4, 5, and 6 hours, than PB platelets. Subsequently, we compared the proliferative effects of CB vs. PB platelet lysates on human primary CB-derived endothelial progenitor cells (EPCs) and amniotic membrane-derived mesenchymal stromal cells (MSCs). Consistent with the effects on HUVEC cells, CB platelet lysates induced approximately 50% more proliferation of EPCs, compared to PB platelet lysates (p<0.001). In contrast, PB platelet lysates were approximately twice more potent in promoting MSCs growth (p<0.001 vs. CB platelets). Since EPCs give rise to the vascular endothelium and MSCs differentiate into mural cells, these results strongly suggested that the AGF content in fetal/neonatal platelets promotes rapid angiogenesis at the expense of a stable vasculature, matching the developmental needs of rapidly growing fetuses and neonates. Finally, since birth is associated with an abrupt change in oxygen concentration (from the profoundly hypoxic intrauterine to the normoxic extrauterine environment), we assessed whether oxygen concentration would modulate the VEGF content in CB-derived cultured MKs (and thus platelets). Consistent with this hypothesis, CB MKs cultured in 5% oxygen released significantly more VEGF in the medium compared to MKs cultured in 20% oxygen (p<0.05). Our findings strongly suggest that the platelet AGF profile changes from neonatal to adult life to help regulate vascular development at different stages of life, and suggest that it might also change in response to environmental factors such as oxygen concentration. Whether a change in platelet angiogenic profile associated with the sudden increase in oxygen concentration during preterm birth contributes to diseases of prematurity characterized by disrupted vascular development (i.e. retinopathy of prematurity) requires further investigation.