Objectives: Platelets contain numerous substances regulating angiogenic response. However, the regulatory role of platelets in blood vessel development remains to be elucidated. We investigated the comprehensive effect of platelets as a cellular system on angiogenesis. Methods: The following approaches were applied: (a) in vitro-aortic ring assay and chemotaxis assay; (b) in vivo-injection of platelet-containing matrigel plug subcutaneously into a mouse followed by immunohistochemical analysis of angiogenic response. Results: Platelets stimulated formation of blood vessels in vitro in the rat aortic ring model via VEGF and bFGF, while blocking of platelet factor-4 promoted this effect. Addition of platelets to the matrigel followed by its subcutaneous injection into a mouse resulted in an intensive migration of fibroblasts into the matrigel as well as formation of blood capillaries de novo. This platelet effect was mediated through bFGF, VEGF, and heparanase. Furthermore, platelet releasate was found to induce endothelial cell chemotaxis. This effect was mediated by a concerted action of intraplatelet bFGF, PDGF, VEGF, and heparanase. Conclusion: Platelets affect different stages of the angiogenic response with a trend to a pro-angiogenic net effect despite the presence of angiogenesis inhibitors such as platelet factor 4. While a concomitant effect of bFGF and VEGF seemed to be essential for the entire process of vessel formation (aortic ring and matrigel models), PDGF and heparanase were effective only at the migration stage.
This article is referred to in the Editorial by A.D. Blann (pages 192–193) in this issue.
The development of new blood vessels is an important process in various physiological and pathological conditions. Excessive angiogenesis underlies pathogenesis of different diseases, such as cancer, atherosclerosis, rheumatoid arthritis, endometriosis, and others . Angiogenesis is regulated by specific cytokines, extracellular matrix fragments and other biologically active molecules, some of which (e.g., VEGF and bFGF) activate angiogenesis, while others (e.g., platelet factor-4 and thrombospondin) exert an inhibitory effect. All these factors, as well as numerous other angiogenic regulators, are present in blood platelets [2–4].
Platelets play a pivotal role in primary hemostasis and comprise the basis for a blood clot at a site of vascular injury where formation of blood capillaries de novo is required. Based on this observation, the important role of platelets in regulating angiogenesis has been suggested by several authors during the last 30 years [5,6]. Platelets have been reported to participate in different clinical conditions in which angiogenesis plays an important role. For example, it was demonstrated that platelets modulate cutaneous  and gastric ulcer healing , and suppress blood–retinal barrier breakdown in diabetic retinal vasculature . A number of platelet cytokines entitled “platelet derived wound healing factors” (PDWHF) were shown to enhance wound healing angiogenesis in rats  and to play a role in the healing process of spinal cord injury . The use of PDWHF for the healing of diabetic ulcers of the lower extremity has reached the level of clinical trials . Platelets play an important role in processes closely related to angiogenesis, such as inflammation and tissue repair (reviewed by Klinger and Jelkmann ). Alongside of clinical observations, experimental data suggest that platelets may affect several stages of the angiogenic response. In particular, platelets were demonstrated to induce human umbilical vein endothelial cells (HUVEC) proliferation and survival, this effect being mediated via FGF-2 and VEGF . Furthermore, platelets were shown to promote tubule formation by HUVEC, which was dependent on platelet adhesion to the endothelial cells . Recent studies demonstrated that, in addition to release of pre-existing compounds, platelets can synthesize new ones upon stimulation due to a large amount of messenger RNA . This fact indicates that platelets may play a previously unknown role in regulating different vascular responses. The complexity of platelet−endothelium interactions is further supported by the finding that VEGF-stimulated endothelial cells, in turn, activate platelets and stimulate platelet adhesion .
Previously published studies concerning platelet effects on angiogenesis dealt mostly with only one separate stage of vessel formation. In contrast, the present study was designed to evaluate the effect of platelets as a cellular system on the angiogenic response in toto using both in vitro and in vivo models. We demonstrate that the effect of angiogenic stimulators prevails in the “balance” between numerous pro- and anti-angiogenic intra-platelet compounds. These results suggest that platelets may play a role in angiogenesis-dependent conditions, such as tumor growth and metastasis on one hand, and response to ischemic event on the other hand.
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).
Bovine aortic endothelial cells (BAEC) were kindly provided by Prof. N. Savion (Tel Aviv University, Israel). These bovine cells respond to human cytokines by intensive proliferation, chemotaxis, and differentiation [18,19], and so far, these cells may be used in experiments with human platelets. All the inhibitors used in this study were anti-human, and they were used in final concentrations two-fold higher than those indicated by the manufacturer as neutralization dose50 (ND50): VEGF receptor tyrosine kinase inhibitor (Calbiochem, San Diego, CA; catalog number 676475)-4 μM; blocking mAbs against bFGF and PDGF (R&D Systems, Minneapolis, MN; catalog numbers AF-233-NA and AB-23-NA, respectively) −0.5 and 10 μg/ml, respectively; neutralizing mAb against PF-4 (Chemicon International, Temecula, CA; catalog number AB1488P) −6 μg/ml. All media, supplements, and matrigel were from Biological Industries (Beit-Haemek, Israel). Recombinant human heparanase and heparanase inhibitor (N-acetylated, glycol-split fragment of heparin) were provided by Prof. I. Vlodavsky (Hadassah Medical Center, Jerusalem). Integrilin was obtained from Key Pharmaceuticals (Kenilworth, NJ). Tirofiban was purchased from Merck (Dartstadt, Germany). Anti-vWF mAb was purchased from Dako (Denmark). Human plasma thrombin, apyrase (from potato, typeGrade I), prostaglandin E1 (PGE1), and crystal violet were from Sigma (Rehovot, Israel).
2.2. Preparation of platelets and platelet releasate
Whole venous blood was drawn from healthy nonsmoking individuals who have been aspirin/NSAID-free for at least 1 month. Blood was collected in vacutainer containing 3.8% citrate. Platelet-rich plasma (PRP) was obtained by centrifugation of whole blood at 120 × g for 12 min. Next, PRP was centrifuged (750 × g, 5 min) in the presence of 5 mM citric acid, the plasma was removed, and the platelet pellet was resuspended in the medium used in the current experiment. Resuspended platelets were immediately added to the appropriate sample.
For preparation of platelet releasate, increasing amounts of pelleted platelets, resuspended in the medium, were treated with 0.5 U/ml thrombin. After 2-min incubation, platelet aggregates were removed by centrifugation at 750 × g for 2 min, supernatant was collected and used in the experiments.
2.3. Aortic ring model
The method described by Nicosia and Ottinetti  was used with some modifications. The thoracic aorta was isolated from Sabra rats (weight 160–190 g, purchased from Harlan Laboratories, Jerusalem, Israel) and immediately placed into warm Bio-MPM medium. Fibroadipose tissue around the vessel was accurately removed by fine microdissecting forceps and scissors. The aorta was sliced into rings 1 mm wide. The rings were than washed three times in sterile warm Bio-MPM medium containing penicillin, streptomycin, and nystatin and embedded in collagen gel obtained from rat tail tendons as described . The collagen solution was prepared by mixing 7 parts of collagen with 1 part of MEM × 10 medium and 2 parts of 0.3 M NaHCO3. The ring-containing collagen gel was prepared in 24-well tissue culture plates in quadruplicate and Bio-MPM (500 μl; supplemented with 1% glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin) was added. Platelets or platelet releasate, with or without blocking mAbs and inhibitory compounds, were added to the medium in the appropriate wells. Wells containing platelets with addition of irrelevant IgG were used as a control. The plates were maintained for 1 week (37 °C, 8% CO2, humidified atmosphere), and the medium, as well as platelets, platelet releasate, and the inhibitors, were changed every 2 days. The use of rat model for studying human-derived compounds was based on our unpublished observations and literature data [22–24], demonstrating that rat aortic rings respond to human cytokines (i.e., VEGF, FGF, endostatin) by intensive angiogenesis.
After the indicated time period, the cultures were fixed with 4% formalin for 24 h and stained with 0.02% crystal violet solution in ethanol (Sigma). The stained samples were photographed with a Nikon (Coolpix 990, Japan) camera connected to the microscope (Olympus CK40, Japan; magnification × 20). Measurement of the area covered by blood vessels (in mm2) was performed using the software ImagePro 4.5 (Media Cybernetics, USA). The results of all experiments (the number of the experiments performed in duplicate is indicated in the corresponding legends) were included in the analysis. The distance from the ring to the edge of the cell front (in millimeters), reflecting both cell proliferation and migration, was assessed by the same software.
2.4. Matrigel plug model
The method described by Passaniti et al.  has been used with minor modification. Briefly, male Sabra mice (7–8 weeks old) were injected subcutaneously at the inter-scapular area with matrigel containing platelets (5 × 108 per 300 μl) with or without mAbs and inhibitors. The mice were kept under regular conditions with standard rodent diet and fresh water ad libitum. After 1 week, the mice were sacrificed by CO2 inhalation and the matrigel plug was removed. After fixation (4% formalin, 24 h), paraffin-embedded sections with the matrigel plugs were prepared and immunostained with mAbs against vWF, an antigen specific for endothelial cells .
Paraffin-embedded matrigel sections (7 μm) were deparaffinized by heating (30 min, 60 °C) and rinsing with xylene (3 × 10 min), and dehydrated by immersing in increasing concentrations of ethanol. Intrinsic peroxidase activity was blocked by H2O2 (3% solution in methanol, 15 min). The samples were treated with pronase (Sigma; 0.1%, 30 min, RT) and then incubated with anti-vWF antibody (1:200, 18 h, 4°C). The staining was performed with Histostain-Plus Kit (Zymed Laboratories, San Francisco, CA) according to the manufacturer's instructions.
2.6. Endothelial cell migration assay
BAEC (generation 10–15) were maintained in DMEM containing 10% FCS until an 80% confluent monolayer was formed. The cells were removed by trypsin–EDTA, washed, counted, and resuspended in DMEM containing 0.1% BSA. Transwell filters (8.0-μm pore size; Corning, USA) were pre-coated with collagen type IV (Sigma; 10 μg/ml) for 1 h at 37 °C. Endothelial cells (2.5 × 104 per 100 μl serum-free medium) were placed in the upper chamber of the transwell system, and platelet releasate with or without inhibitors was added to the lower chamber. The samples were incubated for 18 h (37 °C, 8% CO2, humidified atmosphere). Subsequently, the filters were fixed in 80% cold methanol and stained with propidium iodide (5 mg/ml). Confocal microscopy was performed using a Zeiss LSM-410 confocal laser scanning system attached to a Zeiss Axiovert microscope. Thirty-five optical sections of each filter were made. The samples were compared at section 32, corresponding to the bottom of the filter. A quantitative evaluation of migrated cells, presented in conventional units as a relative part of the “Red” color in the Red–Green–Blue color histogram analysis of the pictures, was performed by Adobe Photoshop.
3.1. Platelets induce angiogenesis in the aortic ring model
The effect of platelets on the angiogenic process was evaluated using the rat aortic ring model. As seen in Fig. 1, platelets induced vessel growth in a dose-dependent manner beginning from concentration as low as 2 × 104 per μl and reaching a plateau at 5 × 105 per μl. A similar dose-dependent pro-angiogenic effect was observed when platelet releasate was used (data not shown). In this case, the minimal concentration of the releasate needed to induce angiogenesis was 5 × 104 per μl platelets. Similarly, the maximal angiogenic response was observed using releasate obtained from 5 × 105 per μl platelets. It should be noted that platelets induced a higher angiogenic response, in absolute values, than platelet releasate.
3.2. Differential effect of platelet cytokines on angiogenesis in the aortic ring model
In order to identify platelet factors responsible for the platelet-induced pro-angiogenic effect, we used blocking mAbs against bFGF, PDGF, and PF-4, as well as VEGF receptor phosporylation inhibitor and synthetic inhibitor of heparanase. As shown in Fig. 2, the VEGF receptor phosporylation inhibitor and anti-bFGF mAbs inhibited platelet-induced angiogenesis. However, it should be noted that such inhibitory effect was demonstrated only in 7 and 6 experiments (out of 10 performed in duplicates) for the VEGF receptor inhibitor and anti-FGF mAb, respectively, whereas in the rest of the experiments, no inhibition was observed (data not shown). Blocking of PDGF or heparanase had no effect in all experiments, whereas blocking of PF-4 resulted in a marked increase in platelet-induced angiogenesis. Irrelevant IgG, added to the medium with platelets, did not have any effect (data not shown).
Thus, blocking of a single platelet pro-angiogenic agent was not always associated with inhibition of platelet-induced angiogenesis. Therefore, we tried to apply combinations of blocking mAbs in the aortic ring system. The results, presented in Fig. 2, show that combined blocking of VEGF receptor phosphorylation, FGF, and PDGF resulted in complete inhibition of angiogenesis in all experiments. Interestingly, the inhibitor and mAbs used did not significantly affect the distance between the ring and the edge of the cell front around the ring, which reflects both cell proliferation and migration (1.2±0.4 mm for platelets only (Fig. 2.2) versus 0.9±0.3 mm for platelets + the inhibitors combination (Fig. 2.6)).
3.3. Role of platelet integrins in platelet-induced angiogenesis
Membrane receptors play a pivotal role in platelet activation and release of the content of alpha-granules. This is true in the aortic ring model as well, although in this model, platelets do not enter into tight contact with endothelial cells. Therefore, we tested the effect of platelet receptor GPIIb-IIIa antagonists on platelet-induced angiogenic response. The experiments performed showed that blocking platelet–platelet interactions via GPIIb-IIIa by either integrilin or tirofiban did not modify vessel development (Fig. 3).
3.4. Effect of platelet activation on their ability to induce angiogenesis
Platelet activation is accompanied by the release of different substances, including those involved in regulation of angiogenesis. Therefore, we activated platelets with thrombin (0.2 U/ml) and studied their effect on vessel growth. As seen in Fig. 3, thrombin-treated platelets induced a markedly higher angiogenic response than intact thrombocytes. Next, platelets were treated with apyrase (2 U/ml), which prevents platelet activation via hydrolyzing released ADP, or with PGE1, platelet antagonist operating via elevation of intraplatelet cAMP level. Apyrase- or PGE1-treated platelets induced significantly weaker angiogenesis as compared to untreated thrombocytes. Neither thrombin nor apyrase/PGE1 alone produced any effect on vessel growth in this model (data not shown). Therefore, platelet activation is important for the promotion of angiogenesis.
3.5. Platelets induce growth factor-dependent angiogenesis in vivo
Next we used an in vivo model of subcutaneous matrigel injection into mice to further evaluate platelet effect on angiogenesis. Staining for vWF showed numerous capillaries and endothelial cells in platelet-enriched matrigel (Fig. 4). This effect was completely blocked by anti-FGF mAb as well as by the VEGF receptor tyrosine kinase inhibitor, whereas blocking of PDGF had no effect. In contrast to the aortic ring, in this model, inhibition of platelet heparanase resulted in a significant decrease in the amount of vessels formed, while inhibition of PF-4 led to a similar upregulation of blood vessel development in the matrigel and the aortic ring model.
3.6. Platelet releasate induces EC migration
Migration of EC to a site of angiogenesis is one of the pivotal steps in the angiogenic process. Therefore, we studied the effect of platelet releasate on EC migration in the transwell system. Analysis performed using confocal microscopy of the EC migrated through the membrane showed that platelet releasate exerted significant stimulation of EC migration, comparable with that induced by 50 ng/ml VEGF (Fig. 5). Separate blocking of VEGF receptor phosphorylation, PDGF, bFGF, or heparanase markedly decreased platelet-induced EC migration. Thus, a concerted action of several intracellular compounds promotes EC migration.
Blood platelets contain numerous substances capable of affecting angiogenesis, such as VEGF, bFGF, PDGF, thrombospondin, endostatin, and PF-4 [27,28]. Moreover, platelets are involved in different processes, that are strictly dependent on new blood vessel development, for example, wound healing . In the present study, we demonstrate that platelets as well as platelet releasate induce angiogenesis in a dose-dependent manner both in vitro and in vivo. Moreover, platelets upregulate such basic stage of the angiogenic process as EC migration.
Due to the large number of pro- and anti-angiogenic compounds stored in platelets, it was important to find out what would be the result of their concerted action. In addition to such potent angiogenesis inducers as VEGF, bFGF, and PDGF, platelets contain strong inhibitors, for example, PF-4 , thrombospondin  and angiostatin . Our experiments showed that the overall platelet effect on angiogenesis is its stimulation. This effect might be mediated either by the platelet granular cytokines released from platelets upon their activation, or by the direct platelet-EC contact through membrane integrins. Of interest was the finding that whole platelets exerted a higher angiogenic response compared to platelet releasate. The method of platelet releasate preparation (see Methods) allows to exclude a significant loss of intraplatelet substances. Furthermore, the design of the aortic ring model, where no contact between platelets and EC occurs, as well as the lack of effect of GPIIb-IIIa inhibitors, suggest that contribution of direct platelet-EC interaction is minimal or absent. Recent studies have demonstrated that platelets contain mRNA of different genes encoding intra-platelet proteins (for example, see reports [33,34]). In light of these results, the higher angiogenic response to platelets than to platelet releasate may probably be explained by additional protein synthesis in platelets, although further studies are needed to investigate this hypothesis.
The studies using the aortic ring model showed that specific blocking of VEGF receptor phosphorylation or bFGF abolishes the platelet-induced angiogenic response. This effect, however, was observed in only part of the experiments. The discrepancy in the effects of anti-cytokine mAbs between different experiments may suggest that a certain difference exists in the ratio of cytokines in the alpha-granules of platelets from different donors. This explanation is further supported by the fact that selective inhibition of the cytokines produced either complete disappearance of vessel growth or no effect at all. An alternative explanation may be that since the platelet cytokines operate in concert, and if none of them prevails, blocking one of them might not necessarily result in downregulation of the total response.
Endothelium injury of different nature is accompanied by inflammation and development of new capillaries . Circulating platelets attached to the vessel wall release various substances to the tissues beyond the injured endothelium . Interestingly, it was recently demonstrated that in vivo implanted platelets, as well as peripheral blood mononuclear cells, migrate towards arterioles and concentrate in perivascular area where angiogenesis occurs . In addition, it is known that platelet releasate contains phospholipids (phosphatidyl serine, phosphatidic acid, lysophosphatidyl ethanolamine and sphingosine-1-phosphate) capable of increasing EC chemotaxis . Here we show that platelet releasate induces EC migration. This finding is in accord with the reported results showing that platelet releasate stimulates the initial recruitment of bone cells to migration . However, in our experiments, the pro-migratory effect was mediated by at least four agents: VEGF, bFGF, PDGF, and heparanase. Interestingly, selective blocking of each molecule resulted in a complete (for VEGF and bFGF) or partial (for PDGF and heparanase) inhibition of EC chemotaxis. This finding suggests that, in contrast to the aortic ring model, each one of the tested regulatory agents is essential for the promotion of endothelial motility, and that a full effect can be reached only in their concerted action. The results, demonstrating the role of PDGF in endothelial migration, are of special interest since the pro-migratory effect of this cytokine has been previously described for smooth muscle cells [39,40]. The exact mechanism of this effect remains to be elucidated. The ability to stimulate EC migration by adhered and activated platelets may be an important mechanism of revascularization in various thrombotic diseases and ischemic conditions (i.e., myocardial infarction).
In conclusion, platelets promote angiogenesis, and substances stored in their granules mediate this effect. While a combined effect of bFGF and VEGF seems to be crucial for the entire process of vessel formation (aortic ring and matrigel models), whereas PDGF and heparanase are effective mainly at the migration stage. Platelet factor-4 exerts an inhibiting effect, although this effect does not seem to overcome the overall stimulatory action of platelets.
This work was supported by the grant of Israeli Academy of Sciences. The authors wish to thank Prof. I. Vlodavsky (Hadassah Medical School, Jerusalem) for the gift of heparanase and heparanase inhibitor, Prof. N. Savion (Tel Aviv University) for providing the bovine aortic endothelial cell line, and Dr. M. Tarshish (Hebrew University, Jerusalem) for the technical assistance.