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Keywords:

  • coagulation;
  • platelet;
  • vascular endothelial growth factor;
  • wound healing

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Summary. Background: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific potent mitogen that induces angiogenesis and microvascular hyperpermeability. Recently, it has been reported that megakaryocytes and platelets contain VEGF in their cytoplasm. Objectives: To elucidate and confirm the bioactivity and role of VEGF in platelets (platelet VEGF), which may be closely related to vascular thrombosis and atherosclerosis. Methods: The VEGF localization in megakaryocytes on bone marrow smears was analyzed by immunofluorescence and confocal laser scanning microscopic analysis. The intracellular VEGF expressed in platelets was determined by flow cytometric analysis. Platelet-rich plasma and washed platelets were used to analyze the secretion of VEGF during platelet aggregation by thrombin or gelatinase A (matrix metalloproteinase-2) stimulation. Immunohistochemical studies for VEGF in the thrombotic region were performed. Results and conclusions: Megakaryocytes and platelets are a very rich source of circulating VEGF. Gelatinase A, which is closely associated with vascular remodeling, enhances the VEGF levels released from platelets. VEGF was clearly detected in the fibrin nets of a thrombus. Taken together, platelet VEGF is bioactive as a direct angiogenic growth factor, and may play a very important role in wound healing and atherosclerosis in conjunction with other platelet cytokines such as platelet-derived growth factor, platelet-derived endothelial cell growth factor, transforming growth factor (TGF)-α, and TGF-β.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Monocytes/macrophages and T-lymphocytes accumulate at sites of endothelial injury or atherosclerosis. This leads to endothelial cell dysfunction; blood flow becomes irregular, and platelet thrombi are formed. In this region, numerous growth-regulatory molecules and cytokines are released from multiple cell types [1], including platelets. Since angiogenesis occurs in the intravascular membrane of atherosclerotic vessels, we considered whether platelets play a role in angiogenesis.

Several growth factors have been reported as potential mediators of the angiogenic process: acidic and basic fibroblast growth factors (aFGF and bFGF) [2,3], platelet-derived growth factor (PDGF) [4,5], platelet-derived endothelial cell growth factor (PD-ECGF) [6], transforming growth factor (TGF)-α and TGF-β[7,8], tumor necrosis factor (TNF)-α[9], angiogenin [10], and interleukin-8 [11]. These factors are active not only on endothelial cells, but also on a wide range of different cell types. Platelets contain PDGF, EGF, TGF-α, and TGF-β, and may play a role in angiogenesis and the repair of injured vessels. Vascular endothelial growth factor (VEGF) [12,13], also called vascular permeability factor (VPF) [14], is an endothelial cell-specific mitogen [15]. The VEGF/VPF (hereafter VEGF) gene in human cells is transcribed into four mRNA species, as a result of alternative splicing. These four isoforms encode protein that is 121, 165, 189, and 206 amino acids in length [16]. The active forms of VEGFs are homodimers [13], and it is known that VEGF is produced by and secreted from various types of cells [17–20], including megakaryocytes and platelets [21,22].

In the present study, we showed that VEGF derived from platelets (platelet VEGF) was bioactive as a direct angiogenic growth factor, and may play an important role in wound healing and atherosclerosis.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Materials

For diagnostic purposes, bone marrow smears from a patient with a non-myeloproliferative disorder were obtained, after informed consent was given. The smears were provided by Dr T. Matsuei (Kameda General Hospital, Chiba, Japan). A paraffin section of an organized thrombus from the right 4th finger was kindly provided by Professor S. Yonezawa (Kagoshima University, Kagoshima, Japan).

ELISA for VEGF

The levels of VEGF in the culture supernatants, serum, and plasma were measured using a colorimetric ELISA as previously described [23].

Preparation of blood samples and platelet aggregation by thrombin or gelatinase A

Peripheral blood was collected with or without 3.8% sodium citrate from 13 healthy volunteers. The blood was centrifuged at 1200 × g for 15 min, and the supernatant from these samples was collected to measure the plasma or serum levels of VEGF. Peripheral blood was collected with 3.8% sodium citrate from nine healthy volunteers. This blood was centrifuged at 160 × g for 15 min to obtain platelet-rich plasma (PRP), and then centrifuged at 1200 × g for 10 min to obtain platelet-poor plasma (PPP). PRP (1 mL) was incubated at 37 °C with a graded series with a final concentration of thrombin (0, 0.5, 1.0, 10.0 U mL−1) (Sigma Chemical Co., St Louis, MO, USA). Washed platelets (1 mL) (7 × 104 µL−1) prepared from PRP in PBS supplemented with 3.8% sodium citrate were stimulated by a graded series of a concentration of gelatinase A (0, 0.2, 2.0 ng mL−1; Yagai Research Center, Yamagata, Japan) for 5 min at 37 °C. The reaction mixtures were centrifuged at 10 000 g in a microcentrifuge (Brinkmann Instruments, Inc., Westbury, NY, USA) for 5 min. The levels of VEGF were measured in the supernatant solution.

Flow cytometry analysis

The platelets obtained from the PRP were washed twice in PBS, and half of the platelets were permeabilized with 70% ethanol for 30 min. Half of them were used as a control, without 70% ethanol treatment. The platelets were then incubated with saturating concentrations of primary antibodies, including monoclonal antihuman VEGF antibody (MV833) [24], for 30 min at 4 °C. After washing, quantitative analysis was performed by flow cytometry using a Cytron absolute (Ortho-Clinical Diagnotics Systems, Tokyo, Japan). A FITC-labeled non-immune mouse antibody was used as a negative control to verify the staining specificity of the experimental antibody.

Immunofluorescence and confocal laser scanning microscopic analysis

The human bone marrow smears were washed twice with PBS and then fixed in 10% formaldehyde buffer at room temperature for 1 h, or fixed with 50% acetone/50% methanol/0.01% Triton X-100 at − 20 °C for 20 min. Each slide was treated with 1% bovine serum albumin (BSA) and then incubated for 1 h at room temperature with a monoclonal antihuman VEGF antibody (MV833) (dilution 1 : 75). Immunohistochemical staining was carried out with FITC-labeled goat antimouse IgG (Sigma) (dilution 1 : 200) for 30 min at room temperature. Negative control samples were treated with non-immune mouse IgG1 as a primary antibody. Confocal laser scanning microscopy (Leica True Confocal Scanner 4D; Leica Lasertechnik GmbH, Heidelberg, Germany) was utilized to examine the findings from the stains more clearly.

Immunohistochemistry

The paraffin sections of thrombus were stained by a standard ABC (streptavidin–biotin–peroxidase complex) immunoperoxidase technique (Vectastain Elite ABC kit; Vector Labs, Inc., Burlingame CA, USA). The tissue sections were incubated for 2 h at room temperature with a monoclonal antihuman VEGF antibody (MV833) (dilution 1 : 50). They were visualized by light microscopy after the diaminobenzidene reaction (Kirkegaard & Perry Labs, Inc., Gaithersburg, MD, USA), staining with 0.2% methyl green, and dehydration.

Statistical analysis

Results are expressed as means ± standard error (SE) of the mean from at least three independent experiments, and each experiment gave similar results. Statistical analysis was performed using Student's t-test, and P < 0.05 was considered statistically significant.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

VEGF concentrations in plasma and serum

The plasma and serum from the same individuals were examined as a pair. The VEGF concentrations in the 13 serum samples (113 ± 18.2 pg mL−1) were significantly (P < 0.0001) higher than those in the 13 plasma samples (10.2 ± 0.87 pg mL−1) (Fig. 1).

image

Figure 1. Vascular endothelial growth factor (VEGF) concentration in the plasma and serum from normal volunteers. Peripheral blood was collected from 13 healthy volunteers with or without citrate anticoagulant. The plasma and serum from the same individuals were examined as a pair. The concentration of VEGF was measured by ELISA. The mean values and SD of the VEGF in the plasma and serum were 10.2 ± 0.87 and 113.8 ± 18.2 pg mL−1, respectively.

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Immunofluorescence analysis of human bone marrow smears

We investigated the VEGF expression in normal megakaryocytes by performing immunofluorescent analysis on human bone marrow smears using confocal laser scanning microscopy. As shown in Fig. 2A, the VEGF antigen was detected diffusely in the cytoplasm of the megakaryocytes. The megakaryocytes were not stained by control mouse IgG1 (Fig. 2B).

image

Figure 2. Indirect immunofluorescent analysis of human bone marrow smears using confocal laser scanning microscopy. (A) Vascular endothelial growth factor (VEGF) antigen was detected diffusely in the cytoplasm of megakaryocytes stained by monoclonal antihuman VEGF antibody (MV833) as the primary antibody. (B) The megakaryocytes were not stained by control non-immune mouse IgG1 as the primary antibody.

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Secretion of VEGF during platelet aggregation

We measured the level of VEGF in the supernatant from activated platelets. As shown in Fig. 3A, the supernatant after aggregation of the platelets in the PRP induced by thrombin contained higher levels of VEGF than PPP. About 14 pg of VEGF was released from 1 × 106 of platelets upon stimulation by 1 U mL−1 thrombin.

image

Figure 3. Release of vascular endothelial growth factor (VEGF) from aggregated platelets. (A) After platelet aggregation was induced by thrombin (0.0–10.0 U mL−1), the VEGF in the supernatant was measured by colorimetric ELISA [23]. (B) Washed platelets were prepared from platelet-rich plasma (PRP), stimulated with gelatinase A (0.0–2.0 ng mL−1), and then incubated for 5 min at 37 °C. The reaction mixtures were centrifuged, and the levels of VEGF in the supernatants were measured [23]. The supernatant of the PRP sample with no thrombin (A), or with no gelatinase A (B) is actually the same as platelet-poor plasma.

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Recently, it has been reported that human platelets express gelatinase A (matrix metalloproteinase-2), and that its release affects platelet aggregation [25]. Therefore, we investigated the effect of gelatinase A on the VEGF levels released from platelets. As shown in Fig. 3B, gelatinase A increased the VEGF levels released from platelets. Gelatinase A produced a maximal response at a concentration of 2.0 ng mL−1.

Intracellular VEGF expression in platelets

We analyzed the expression of VEGF in platelets by flow cytometric analysis. VEGF staining was very weak on the cell surface of non-permeabilized platelets (Fig. 4A), while a high intensity of VEGF staining was observed in the cytoplasm of permeabilized cells by 70% ethanol (Fig. 4B). These results demonstrate that platelets contain VEGF in their cytoplasm but not in their cell membrane.

image

Figure 4. Intracellular platelet vascular endothelial growth factor (VEGF) expression. The levels of expression of VEGF were analyzed quantitatively by flow cytometric analysis. The black area indicates cells treated with a monoclonal antihuman VEGF antibody (MV833), whereas the white area indicates cells treated with control non-immune mouse IgG1 as the primary antibody. (A) The VEGF was very slightly expressed on the cell surface of the non-permeabilized platelets. (B) A high intensity of VEGF staining was observed in the cytoplasm of permeabilized platelets by 70% ethanol.

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VEGF expression in a thrombus

We performed immunohistochemical studies of a thrombus using the ABC method to investigate the function of the VEGF released from platelets. In the thrombotic region of the right 4th finger, the fibrin nets (Fig. 5A) were clearly stained using a monoclonal antihuman VEGF antibody (MV833) [24] (Fig. 5B). The same area was not stained by a control mouse IgG1 (data not shown). These data strongly suggest that the VEGF released from platelets included in these fibrin nets may be stained.

image

Figure 5. Vascular endothelial growth factor (VEGF) expression in a thrombus. (A) Fibrin nets were observed in the thrombus (H&E stain, × 780). (B) The same area was stained using a monoclonal antihuman VEGF antibody (MV833) (immunoperoxidase stain, × 780). This area was not stained by the control mouse IgG1 (data not shown).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

In vascular injury, the endothelial cells are damaged, and platelets aggregate at the damaged sites. Platelets release PDGF, which induces smooth muscle cells to proliferate [26]. They may also produce VEGF, which induces the migration and angiogenesis of endothelial cells. We confirmed that PDGF and TGF-β are released from platelets with VEGF upon stimulation by thrombin (data not shown). Activated macrophages and fetal human aortic smooth muscle cells have also been reported to express VEGF mRNA [12,19]. Recently, Maloney et al. [27] reported that platelets release VEGF upon stimulation by thrombin. We also confirmed, by immunofluorescence confocal laser scanning microscopic analysis (Fig. 2) and flow cytometry analysis (Fig. 4), that the VEGF level in the serum was much higher than in plasma and platelets that have VEGF in their cytoplasm. There have been several reports describing how platelets increase vascular permeability [28–30]. Miyazono et al. [31] reported that gel exclusion chromatography showed a low-molecular-weight protein which activates the proliferation of endothelial cells, and is not PDGF or platelet-derived endothelial cell growth factor (PD-ECGF) [32]. We think that this protein in platelets and VEGF may be identical.

As recently reported, gelatinase A (MMP-2) may mediate a new pathway of platelet aggregation [25]. Gelatinase A belongs to the group of matrix metalloproteinases that are involved in remodeling the extracellular matrix. Gelatinase A is secreted by human platelets [33] as well as human epithelial cell lines [33,34], fibroblasts [35,36], endothelial cells [37], and macrophages [38]. We demonstrated that gelatinase A enhanced the VEGF levels released from platelets (Fig. 3). This suggests that in vascular injury or thrombosis, gelatinase A may be released at the time of platelet aggregation and may synergize with VEGF.

In thrombi, the basic structure is formed by a fibrin mesh in which blood cells are trapped. In fibrin gels, there is rapid vascularization [39]. In this study, we detected VEGF in the fibrin nets of a thrombus. Our conclusion is that vascular injury and platelet activation may result in VEGF release from the platelets, which acts as an angiogenic factor.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Portions of this work were presented at the XVIth Congress of the International Society on Thrombosis and Haemostasis. Florence, Italy, 1997. This study was supported by grants from the Neuroimmunological Disease Research Committee of the Ministry and Welfare of Japan, Grant-in-Aid for Scientific Research (11470147 and 13670659) of the Ministry of Education, and the Mitsubishi Pharmaceutical Research Foundation. We thank Dr Takao Matsuei, Kameda General Hospital, for providing the bone marrow smears, and Professor Suguru Yonezawa, Kagoshima University, for providing the paraffin sections of a thrombus. We also thank Miss Nobue Uto and Miss Hisayo Sameshima for their technical assistance.

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  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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