Development of improved strategies for therapeutic angiogenesis remains an imperative task for treatment of a variety of pathological conditions. Of all the circulating blood cells, platelets are the most abundant sources of endogenous angiogenic regulators. In this study we challenged the hypothesis that combination of platelet-derived factors with extracellular nucleotides, also known as regulators of the vascular growth, would have even more potent angiogenic effects in vitro and in vivo. Our data demonstrated that platelet extracts and extracellular adenine nucleotides exerted similar, but not identical effects on endothelial proliferation, migration and tube formation. The main conclusion from this study is that extracellular adenine nucleotides modulate angiogenic effects of platelet extracts in vitro and, importantly, lead to a greater amount of functional capillary formation in Matrigel plugs in vivo.
As mentioned above, platelets contain multiple constituents including angiogenic mediators, extracellular matrix components, nucleotides, sugars, growth factors, antimicrobial peptides, and cytokines that can support wound healing and angiogenesis [6–8, 12, 14, 30, 33]. Angiogenic effects of platelets have been previously demonstrated in several model systems. Recent reports have shown some examples that platelet rich plasma (PRP) and platelets can substitute for animal serum as a nutrient source for human cells in culture and therefore, may have the capacity to provide nourishment or survival factors to cells at risk from the compromised vascular supply at a wound site [[37, 38] and Mark Roedersheimer, unpublished observation]. It was demonstrated, that a potent chemotactic and angiogenic activity is generated by factors released from activated platelets [7, 9, 30]. The presence of platelets not only stimulates vessel growth but also plays a critical role in preventing hemorrhage from the angiogenic vessels  supporting a theory of functional link between hemostasis and angiogenesis.
However, despite a strong evidence of angiogenic effects of platelets, there is also some degree of skepticism about the utility of platelet-derived components for a modulation of angiogenesis due to a high degree of variability in clinical outcomes, which are probably associated with: (i) lack of sufficient release of key growth factors following an incomplete platelet "activation" with a single factor, such as thrombin, during PRP preparation; (ii) failure to separate the platelet-derived growth factors from the bulk of the highly concentrated serum components, which may limit bioavailability; (iii) failure to determine at least the protein concentration of the active platelet extract (separated from the plasma) and using this to establish a proper dosing; and iv) variability in the composition, and therefore activity, of platelet extracts from different individuals that may need to be accounted for in the dosing. Reducing variability in the clinical response to PRP remain as important goal in bringing platelet extract preparation methods to full utility.
In the present study we demonstrated that total cell extracts isolated from preserved platelets have an angiogenic potential. Our approach of using total platelet extracts assumed that a crude extract isolated by a lysis of platelet pellet, even though lacking the ability of intact platelets to sense and respond to signals in a tissue environment, can still be used to produce a balanced regenerative response in a tissue. According to our established procedure, soluble platelet extracts were prepared by platelet sonication and subsequent stabilization of soluble components through the dialysis of cellular extract against 10 mM HCl in a 3.5 KDa cut-off membrane. Based on some reports, mildly acidic environment (also typical for wound sites) may possibly optimize the stability and activity of platelet-derived factors such as PDGF and TGFβ [39, 40]. However, the dialysis step removes small molecular weight soluble components, including nucleotides and amino acids from the fraction of growth factors and cytokines. Therefore, it can be assumed that when platelet extract preparation includes a dialysis step, supplementation (or "reconstruction") of dialyzed platelet extracts with exogenous adenine nucleotides could modulate or potentially enhance the angiogenic effects of platelet-derived extracts.
A variety of pathological conditions in the vasculature is accompanied with elevated levels of extracellular ATP and ADP, platelet immobilization, activation, and their adhesion to endothelial cells, with ultimate endothelial cell activation. These events may possibly lead to angiogenesis and initiation of vascular regeneration processes [23, 41–44]. Activated platelets release factors capable of promoting hematopoietic stem cell migration into a vascular injury site and their differentiation into endothelial cells, which may favor survival via angiogenic signaling pathways [32, 45]. We expect that extracellular nucleotides released from platelets and endothelial cells could act in concert with multiple growth factors and cytokines. Previously, we demonstrated that extracellular ATP exerts dramatic effects on VVEC mitogenesis, migration and tube formation [21, 23]. However, angiogenic effects of extracellular nucleotides in the in vivo models of angiogenesis, as well as combined effects of extracellular nucleotides and platelet-derived growth factors on angiogenesis have not yet been investigated. Our present study provides new evidence that platelet-derived extracts and adenine nucleotides exerted diverse, but complementary angiogenic effects in isolated VVEC. Platelet extract induced DNA synthesis and affected proliferative responses in VVEC. The proliferative response was at its highest at 48 hrs, followed by a decrease at 60 hrs. This biphasic effect may suggest a possibility that the initial increase in VVEC proliferation may be followed by more long-term regulatory signals leading to endothelial differentiation, stabilization and/or morphological changes. Strikingly, extracellular ATP, despite strong effects on DNA synthesis, did not induce VVEC proliferation and to some extent diminished proliferative responses induced by platelet extracts measured at 48 hrs, again, suggesting prolonged and more complex integrative signaling events underlying the action of extracellular nucleotides and platelet-derived angiogenic factors.
Our study also revealed differences in extracellular ATP- and platelet extract-induced migratory responses. Extracellular ATP had a more potent effect (~75- fold) on VVEC migration in contrast to the effect induced by platelet extracts (~7-20-fold). However, more dramatic effects (up to 65-fold) can be observed in response to platelet extracts derived some units of preserved human platelets (Figure 4B and unpublished observations). Interestingly, in response to extracellular ATP all migrated VVEC adhered to and spread uniformly on the underside of the transwell chamber membrane, whereas in response to platelet extracts, aggregates of migrated cells were observed on the underside of the membrane. We also found that platelet extracts, in a concentration-dependent manner, diminish ATP-induced VVEC migration, which was accompanied by a more dramatic cell clustering on the underside of the chamber membrane. Similar to our findings, a recent study by O'Connor demonstrated "adherent migration" of THP-1 in response to platelet soluble fractions , suggesting that even in the migration assay setting, platelet extracts induce structural and adhesive changes in endothelial cells. This morphogenetic effect of platelet extract was further demonstrated in the Matrigel tube formation assay. We found that platelet extract and extracellular ATP were almost equipotent in the induction of satisfactory-developed tubes on growth factor-reduced Matrigel. In addition, the total number of well-developed tubes was significantly increased when VVEC were co-stimulated with platelet extracts and extracellular ATP. Together, the in vitro studies demonstrated that both extracellular ATP and platelet extracts exert angiogenic effects in VVEC, but only morphogenetic effect was potentiated in response to the combined action of extracellular ATP and platelet extracts. Further investigation is necessary to define molecular mechanisms contributing to intracellular signaling cross-talk initiated by purinergic receptors and receptor tyrosine kinases in VVEC.
Recenty, we demonstrated a predominant involvement of P2Y1 and P2Y13 receptors in Ca2+ and mitogenic response in VVEC . In this study we further investigated a contribution of purinergic receptor subtypes in VVEC angiogenesis. Using an antagonist approach, we demonstrated an involvement of P2Y1 and P2Y13 receptors in ATP-induced DNA synthesis and tube formation, as well as an involvement of P2Y13 receptors in VVEC migration. An inhibitory effects of suramin (non-selective P2Y antagonist) on ATP-induced DNA synthesis and tube formation may also suggest a contribution of P2Y2 receptors in these angiogenic responses. In addition, potent inhibitory effects of DIDS on DNA synthesis, migration, and, to a lower extent, tube formation may suggest an involvement of P2X receptors in mediating nucleotide signaling in VVEC. We also found, that some angiogenic effects of platelet extract, although to a different degree, can be attenuated by purinergic receptor antagonists (suramin and MRS2179). Whether this can be explained by the presence of residual endogenous nucleotides or other purinergic receptor ligands (such as cysteinyl leukotrienes) in platelet extracts remains to be determined and represents an interesting possibility for future investigations.
Consistent with the results from the in vitro studies, platelet-derived extracts displayed a dose-dependent angiogenic response in the Matrigel plug assay in 50 ICR mice. The effect was evaluated in terms of the number of tube-like structures, as well as functional capillaries containing red blood cells sufficiently far from a native tissue boundary to exclude their preexistence. We have previously shown that even very low concentrations of platelet-derived extracts (<50 μg/ml) speeded up the healing process of incision wounds placed on the backs of mice (unpublished observation). However, some variations in minimal effective concentration of platelet extracts were observed among human plasma sources. For instance, platelet extracts used in this study exhibit the highest activity at concentrations between 16 and 64 μg/ml. The functionality of capillaries in Matrigel plugs was evaluated based on microscopic detection of erythrocytes within the capillary lumen, suggesting a link to the systemic circulation. In agreement with the in vitro data, we observed increased formation of tube-like structures in Matrigel plugs supplemented with platelet extract. Despite a number of studies showing angiogenic effects of extracellular nucleotides in various cell systems, the angiogenic effects of extracellular nucleotides in the in vivo Matrigel plug model have not been previously investigated. To our surprise, tube formation in the Matrigel plugs supplemented with extracellular ATP was even lower than the tube formation in the control plugs.
It would have been expected that extracellular ATP might be hydrolyzed to adenosine by endothelial and blood cell ecto-nucleotidases, resulting in decreased plug vascularization. Therefore, we evaluated the effect non-hydrolyzable nucleotide analogs, ATPγS, ADPβS, MeSATP, MeSADP. No additional tube formation was observed in response to these nucleotides, suggesting that decreased plug vascularization in response to ATP could not be explained by its hydrolysis to adenosine. Remarkably, we found more cellularity in some Matrigel plugs, containing mixtures of high concentrations of extracellular nucleotides (1 mM). Considering that monocytes/macrophages and endothelial progenitor cells may contribute to neovascularization, and can be attracted by platelet-derived microparticle components [32, 46–49], we speculate that inflammatory and/or progenitor cells may also contribute to angiogenic responses in Matrigel plugs. This possibility will be further investigated in our laboratory. Histological evaluation of Matrigel plugs revealed that both immature tubular structures and functional capillaries were formed. Although tube formation in the Matrigel plugs, supplemented with extracellular ATP was even lower than the tube formation in the control plugs, higher numbers of functional capillaries and PECAM -positive cells were observed in ATP and nucleotide-containing Matrigel plugs compared to control. In some plugs, blood infiltrates were found in addition to well-formed capillary structures, indicating that newly-formed vessels could be, at least in part, immature and leaky. In the meantime, the amount of functional capillaries and PECAM-positive cells was even higher in Matrigel plugs containing platelet extracts and nucleotides (compared to Matrigel plugs containing either nucleotide or platelet extract). These observations are consistent with the idea that extracellular nucleotides and platelet -derived growth factors provide complementary regulatory signals necessary to promote neovessel growth and maturation.