Dear Editor,

We read the paper titled “Anti-permeability and anti-proliferative effects of standard and frozen bevacizumab on choroidal endothelial cells” by Peters et al [1] in Br J Ophthalmol. Dec. 2006 issue with great interest. There are a number of issues that we would like to point out with regards to the methodology. The authors have solely used scratch assay in the presence of 50ng/ml VEGF +- 0.5mg/ml bevacizumab to assess cell proliferation. They demonstrate that when bevacizumab was added wound closure was retarded (data presented in figures 4 and 5) and they infer that this is because of antiproliferative effect of bevacizumab on choroidal endothelial cells. We would like to point out that scratch assay is an accepted method for assessing cell migration,[2-4] but it is not a method for assessing cell proliferation. There are several limitations in using this assay for estimating cell proliferation. If proliferating cells fail to migrate, the wound will not close. On the other hand, the migrating cells can close the wound without undergoing proliferation. Therefore, based on this evidence the authors can not draw conclusions regarding proliferation of cells. The authors have cited Roberts and Palade [5] for the proliferation assay used in this paper. However, the cited paper has not used any such method for assessing cell proliferation. Additionally, in figure 5, the authors have provided microscopic evidence for the cell proliferation assay they have used. In the micrographs provided it appears that the cell density in bevacizumab treated sample is much less than sample treated with VEGF alone that would add additional bias. With regards to proliferation assay, there are excellent methods available to do this in cell cultures.[6-8] These methods utilize Bromodeoxyuridine, tritiated thymidine and carboxyl fluorescent succinimidyl ester. There appears to be no apparent limitation why these methods could not be employed, especially in a paper wherein proliferation is a major focus.

Sincerely,

Rajesh K Sharma, M.D., Ph.D.

Kakarla V. Chalam, M.D., Ph.D.

Department of Ophthalmology

University of Florida Health Science Center

Jacksonville, FL

References

1. Peters S, Julien S, Heiduschka P et al. Anti-permeability and anti-proliferative effects of standard and frozen bevacizumab on choroidal endothelial cells. Br.J.Ophthalmol. 2006.

2. Johnson DA, Fields C, Fallon A et al. Polyamine-dependent migration of retinal pigment epithelial cells. Invest Ophthalmol.Vis.Sci. 2002;43:1228-33.

3. Theisen CS, Wahl JK, III, Johnson KR et al. NHERF Links the N- Cadherin/Catenin Complex to the PDGF Receptor to Modulate the Actin Cytoskeleton and Regulate Cell Motility. Mol.Biol.Cell 2007.

4. Soderholm J, Heald R. Scratch n' screen for inhibitors of cell migration. Chem.Biol. 2005;12:263-5.

5. Roberts WG, Palade GE. Increased microvascular permeability and endothelial fenestration induced by vascular endothelial growth factor. J.Cell Sci. 1995;108 ( Pt 6):2369-79.

6. Luzyanina T, Mrusek S, Edwards JT et al. Computational analysis of CFSE proliferation assay. J.Math.Biol. 2007;54:57-89.

7. Nakamura T, Ang LP, Rigby H et al. The use of autologous serum in the development of corneal and oral epithelial equivalents in patients with Stevens-Johnson syndrome. Invest Ophthalmol.Vis.Sci. 2006;47:909-16.

8. Ye L, Haider HK, Jiang S et al. High efficiency transduction of human VEGF165 into human skeletal myoblasts: in vitro studies. Exp.Mol.Med. 2003;35:412-20.