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Communications to the Editor |

Is Measurement of Serum Vascular Endothelial Growth Factor Reliable in Patients With Active Pulmonary Tuberculosis? FREE TO VIEW

Elisa Arena, MD; Simone Ferrero, MD
Author and Funding Information

Affiliations: University of Genoa, Genoa, Italy,  Osmangazi University Medical School, Eskisehir, Turkey

Correspondence to: Simone Ferrero, MD, Department of Obstetrics and Gynaecology, San Martino Hospital, University of Genoa, Largo R. Benzi 1, 16132, Genoa, Italy; e-mail: simone.ferrero@fastwebnet.it



Chest. 2005;127(2):686-687. doi:10.1378/chest.127.2.686
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To the Editor:

In a recent issue of the CHEST (June 2004), Alatas et al1 measured serum vascular endothelial growth factor (VEGF) concentrations in subjects with active pulmonary tuberculosis, and compared the changes in concentration before and after therapy. We would like to bring to your attention some methodological aspects of this study that might have caused relevant bias in the conclusion.

It is well-known that VEGF resides in the α-granules of platelets and is released during blood clotting.2Therefore, serum VEGF level is not a reliable indicator of the circulating extracellular VEGF levels in vivo, and it principally reflects platelet count rather than VEGF synthesis by peripheral tissues. The authors reported that serum VEGF levels are increased in patients with active pulmonary tuberculosis when compared with those of subjects with inactive tuberculosis and those of healthy subjects. Thrombocytosis is common in subjects with active pulmonary tuberculosis, and it has been shown that, in these patients, platelets aggregate excessively in vitro.5 As a consequence, we think that the higher serum VEGF levels observed by the authors in the patients with active pulmonary tuberculosis were caused by increased platelet counts and increased platelet degranulation in vitro rather than by the fact that the “inflammatory mass stimulates the production of VEGF in order to supplant the needed blood supply by the formation of new blood vessels,” as suggested by the authors. Our hypothesis is supported by the authors’ observation that VEGF levels decrease after treatment when platelet count typically declines.,5Considering that a direct correlation between platelet count and serum VEGF level has been described,6it is advisable to correct VEGF serum levels for variations in platelet count.7

Furthermore, it must be pointed out that the authors did not report the length of time between venipuncture and the separation of serum from blood cells. This interval should always be standardized and declared because serum VEGF concentrations increase in a time-dependent manner.8 In particular, allowing the whole-blood sample to clot for between 2 and 6 h before serum is collected may reduce the time-dependent, nonuniform release of VEGF.6

In plasma, platelet degranulation is minimized by adding anticoagulants to blood samples, and, as a consequence, plasma VEGF concentrations are up to 20 times lower than the matched serum VEGF concentrations.8Citrate/theophylline/adenosine/dipyridamole plasma should be used when circulating extracellular VEGF levels are measured.9

In conclusion, we think that the increase in serum VEGF levels observed by the authors in subjects with active pulmonary tuberculosis was caused by higher platelet counts and enhanced platelet degranulation in vitro, and does not reflect increased circulating extracellular VEGF levels in vivo. A meticulous standardization of blood sampling is mandatory when serum VEGF levels are measured. Serum VEGF concentrations should always be corrected for the variations in platelet count. Finally, it would be interesting to confirm the results reported by the authors with citrate/theophylline/adenosine/dipyridamole plasma samples.

Alatas, F, Alatas, O, Metintas, M, et al (2004) Vascular endothelial growth factor levels in active pulmonary tuberculosis.Chest125,2156-2159. [CrossRef] [PubMed]
 
Wartiovaara, U, Salven, P, Mikkola, H, et al Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.Thromb Haemost1998;80,171-175. [PubMed]
 
Baynes, RD, Bothwell, TH, Flax, H, et al Reactive thrombocytosis in pulmonary tuberculosis.J Clin Pathol1987;40,676-679. [CrossRef] [PubMed]
 
Robson, SC, White, NW, Aronson, I, et al Acute-phase response and the hypercoagulable state in pulmonary tuberculosis.Br J Haematol1996;93,943-949. [CrossRef] [PubMed]
 
Turken, O, Kunter, E, Sezer, M, et al Hemostatic changes in active pulmonary tuberculosis.Int J Tuberc Lung Dis2002;6,927-932. [PubMed]
 
Werther, K, Christensen, IJ, Nielsen, HJ Determination of vascular endothelial growth factor (VEGF) in circulating blood: significance of VEGF in various leucocytes and platelets.Scand J Clin Lab Invest2002;62,343-350. [CrossRef] [PubMed]
 
Gunsilius, E, Petzer, A, Stockhammer, G, et al Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood.Oncology2000;58,169-174. [CrossRef] [PubMed]
 
Banks, RE, Forbes, MA, Kinsey, SE, et al Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology.Br J Cancer1998;77,956-964. [CrossRef] [PubMed]
 
Wynendaele, W, Derua, R, Hoylaerts, MF, et al Vascular endothelial growth factor measured in platelet poor plasma allows optimal separation between cancer patients and volunteers: a key to study an angiogenic markerin vivo?Ann Oncol1999;10,965-971. [CrossRef] [PubMed]
 
To the Editor:

We read with interest Dr. Ferrero’s comments regarding our recently published work (June 2004)1about vascular endothelial growth factor (VEGF) levels in tuberculosis patients. We disagree, however, with his conclusion that for the accurate measurement of circulating VEGF levels, measuring serum levels is completely unsuitable. Indeed, VEGF expression has been found not only in platelets, but also in activated macrophages, neutrophils, smooth muscle cells, monocytes, and T lymphocytes. However, the extent to which these cells contribute to the circulating VEGF levels in vivo, and its physiologic and pathologic relevance is not yet clear. In a recent article, Mittermayer et al2 showed that systemic plasma concentrations of VEGF are increased by inflammation that is independent from thrombin formation but is associated with neutrophil degranulation. They proposed that the activation of leukocytes rather than platelets plays a role in the marked increase in VEGF levels. In another view, activated platelets have been found to release VEGF together with β-thromboglobulin, suggesting that VEGF resides in the α-granules of the platelets.3 This finding leaves open the possibility that at least part of the VEGF in platelets has come from plasma by endocytosis.

Dr. Ferrero incorrectly stated that thrombocytosis is common in patients with active pulmonary tuberculosis. Thrombocytosis is not common in patients with active pulmonary tuberculosis. Recently, Olaniyi and Aken’Ova4observed thrombocytosis only in 12.9% of their patients with active pulmonary tuberculosis. Other articles by Morris5and Akintunde et al6 have observed thrombocytosis in 33% and 18%, respectively, of their pulmonary tuberculosis patients. We observed thrombocytosis in only 11% of pulmonary tuberculosis patients. Moreover, we found thrombocytosis in only 1 of 10 patients with active pulmonary tuberculosis in whom we were able to examine serum VEGF levels at the end of tuberculosis treatment. Thrombocytosis is a consequence of the general inflammatory state of the body against the infection. Also, VEGF is known to be involved in inflammation and wound healing. Therefore, in our view it is hard to explain the increase of serum VEGF levels in patients with active pulmonary tuberculosis by using thrombocytosis alone.

The second important issue raised by Dr. Ferrero is the standardization of the sampling process. In our study setting, all samples were drawn and processed by the same biochemist. The separation of sera from blood cells was performed in 30 to 45 min.

Few studies in the literature710 concerning VEGF levels in tuberculosis patients have determined serum VEGF levels. Moreover, intense angiogenesis was shown ultrastructurally in active pulmonary tuberculosis lesions.11 The expression of VEGF in alveolar macrophages around active tuberculosis lesions was shown by immunohistochemistry.7

Finally, the percentage of thrombocytosis present in our active pulmonary tuberculosis patients is not high, and the sample preparation procedures are well-designed and standardized. Although the type of sample that should be used in VEGF measurements is still a matter of debate, all studies regarding VEGF levels in pulmonary tuberculosis patients were performed using serum samples. However, further research is needed to compare the relationship of VEGF levels in serum and plasma, and different anticoagulant agents, since the existing studies comparing serum and plasma VEGF levels were performed with cancer patients.

References
Alatas, F, Alatas, O, Metintas, M, et al Vascular endothelial growth factor levels in active pulmonary tuberculosis.Chest2004;125,2156-2159. [CrossRef] [PubMed]
 
Mittermayer, F, Pleiner, J, Schaller, G, et al Marked increase in vascular endothelial growth factor concentrations duringEscherichia coliendotoxin-induced acute inflammation in humans.Eur J Clin Invest2003;33,758-761. [CrossRef] [PubMed]
 
Wartiovaara, U, Salven, P, Mikkola, H, et al Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.Thromb Haemost1998;80,171-173. [PubMed]
 
Olaniyi, JA, Aken’Ova, YA Haematological profile of patients with pulmonary tuberculosis in Ibadan, Nigeria.Afr J Med Med Sci2003;32,239-242. [PubMed]
 
Morris, CD The radiography, haematology and biochemistry of pulmonary tuberculosis in the aged.Q J Med1989;71,529-536. [PubMed]
 
Akintunde, EO, Shokunbi, WA, Adekunle, CO Leucocyte count, platelet count and erythrocyte sedimentation rate in pulmonary tuberculosis.Afr J Med Med Sci1995;24,131-134. [PubMed]
 
Matsuyama, W, Hashiguchi, T, Matsumuro, K, et al Increased serum levels of vascular endothelial growth factor in pulmonary tuberculosis.Am J Respir Crit Care Med2000;162,1120-1122. [PubMed]
 
Abe, Y, Nakamura, M, Oshika, Y, et al Serum levels of vascular endothelial growth factor and cavity formation in active pulmonary tuberculosis.Respiration2001;68,496-500. [CrossRef] [PubMed]
 
Ziora, D, Sielska-Spytek, E, Dwomiczak, S, et al VEGF (vascular endothelial growth factor) concentration in serum and pleural fluid of patients with pleural malignancy and pleural tuberculosis.Pneumonol Alergol Pol2002;70,458-467. [PubMed]
 
Hamed, EA, El-Noweihi, AM, Mohammed, AZ, et al Vasoactive mediators (VEGF and TNF-alpha) in patients with malignant and tuberculous pleural effusions.Respirology2004;9,81-86. [CrossRef] [PubMed]
 
Ridley, MJ, Heather, CJ, Brown, I, et al Experimental epithelioid cell granulomas, tubercle formation and immunological competence: an ultrastructural analysis.J Pathol1983;141,97-112. [CrossRef] [PubMed]
 

Figures

Tables

References

Alatas, F, Alatas, O, Metintas, M, et al (2004) Vascular endothelial growth factor levels in active pulmonary tuberculosis.Chest125,2156-2159. [CrossRef] [PubMed]
 
Wartiovaara, U, Salven, P, Mikkola, H, et al Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.Thromb Haemost1998;80,171-175. [PubMed]
 
Baynes, RD, Bothwell, TH, Flax, H, et al Reactive thrombocytosis in pulmonary tuberculosis.J Clin Pathol1987;40,676-679. [CrossRef] [PubMed]
 
Robson, SC, White, NW, Aronson, I, et al Acute-phase response and the hypercoagulable state in pulmonary tuberculosis.Br J Haematol1996;93,943-949. [CrossRef] [PubMed]
 
Turken, O, Kunter, E, Sezer, M, et al Hemostatic changes in active pulmonary tuberculosis.Int J Tuberc Lung Dis2002;6,927-932. [PubMed]
 
Werther, K, Christensen, IJ, Nielsen, HJ Determination of vascular endothelial growth factor (VEGF) in circulating blood: significance of VEGF in various leucocytes and platelets.Scand J Clin Lab Invest2002;62,343-350. [CrossRef] [PubMed]
 
Gunsilius, E, Petzer, A, Stockhammer, G, et al Thrombocytes are the major source for soluble vascular endothelial growth factor in peripheral blood.Oncology2000;58,169-174. [CrossRef] [PubMed]
 
Banks, RE, Forbes, MA, Kinsey, SE, et al Release of the angiogenic cytokine vascular endothelial growth factor (VEGF) from platelets: significance for VEGF measurements and cancer biology.Br J Cancer1998;77,956-964. [CrossRef] [PubMed]
 
Wynendaele, W, Derua, R, Hoylaerts, MF, et al Vascular endothelial growth factor measured in platelet poor plasma allows optimal separation between cancer patients and volunteers: a key to study an angiogenic markerin vivo?Ann Oncol1999;10,965-971. [CrossRef] [PubMed]
 
Alatas, F, Alatas, O, Metintas, M, et al Vascular endothelial growth factor levels in active pulmonary tuberculosis.Chest2004;125,2156-2159. [CrossRef] [PubMed]
 
Mittermayer, F, Pleiner, J, Schaller, G, et al Marked increase in vascular endothelial growth factor concentrations duringEscherichia coliendotoxin-induced acute inflammation in humans.Eur J Clin Invest2003;33,758-761. [CrossRef] [PubMed]
 
Wartiovaara, U, Salven, P, Mikkola, H, et al Peripheral blood platelets express VEGF-C and VEGF which are released during platelet activation.Thromb Haemost1998;80,171-173. [PubMed]
 
Olaniyi, JA, Aken’Ova, YA Haematological profile of patients with pulmonary tuberculosis in Ibadan, Nigeria.Afr J Med Med Sci2003;32,239-242. [PubMed]
 
Morris, CD The radiography, haematology and biochemistry of pulmonary tuberculosis in the aged.Q J Med1989;71,529-536. [PubMed]
 
Akintunde, EO, Shokunbi, WA, Adekunle, CO Leucocyte count, platelet count and erythrocyte sedimentation rate in pulmonary tuberculosis.Afr J Med Med Sci1995;24,131-134. [PubMed]
 
Matsuyama, W, Hashiguchi, T, Matsumuro, K, et al Increased serum levels of vascular endothelial growth factor in pulmonary tuberculosis.Am J Respir Crit Care Med2000;162,1120-1122. [PubMed]
 
Abe, Y, Nakamura, M, Oshika, Y, et al Serum levels of vascular endothelial growth factor and cavity formation in active pulmonary tuberculosis.Respiration2001;68,496-500. [CrossRef] [PubMed]
 
Ziora, D, Sielska-Spytek, E, Dwomiczak, S, et al VEGF (vascular endothelial growth factor) concentration in serum and pleural fluid of patients with pleural malignancy and pleural tuberculosis.Pneumonol Alergol Pol2002;70,458-467. [PubMed]
 
Hamed, EA, El-Noweihi, AM, Mohammed, AZ, et al Vasoactive mediators (VEGF and TNF-alpha) in patients with malignant and tuberculous pleural effusions.Respirology2004;9,81-86. [CrossRef] [PubMed]
 
Ridley, MJ, Heather, CJ, Brown, I, et al Experimental epithelioid cell granulomas, tubercle formation and immunological competence: an ultrastructural analysis.J Pathol1983;141,97-112. [CrossRef] [PubMed]
 
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