Tuberculosis. 2013 May;93(3):373-80.

Modulation of angiogenic factor VEGF by DNA-hsp65 vaccination in a murine CNS tuberculosis model.

Fabíola C.R. Zucchi, Ana Maria C. Tsanaclis, Quintino Moura-Dias Jr., Célio L. Silva, Adriana Pelegrini-da-Silva, Luciano Neder, Osvaldo M. Takayanagui

Corresponding author: (F.C.R. Zucchi)



Tuberculosis (TB) is a serious public health problem. Development of experimental models and vaccines are essential to elucidate physiopathological mechanisms and to control the disease. Vascular endothelial growth factor (VEGF) is a potent activator of vascular permeability and angiogenesis. VEGF seems to participate in breakdown of the blood brain-barrier (BBB) in tuberculous meningitis (TBM), contributing to worsening of disease. Therefore, the objective here was to extent the characterization of our previously described murine model of central nervous system TB (CNS-TB) by describing the VEGF participation in the CNS disease, and suggesting a vaccination plan in mice. Plasmid encoding DNA protein antigen DNAhsp65 has been described as a protector against TB infection and was used here to test its effectiveness in the prevention of VEGF production and TB disease. Vaccinated mice and its controls were injected with Mycobacterium bovis bacillus Calmette-Guerin (BCG) in cerebellum. Four weeks after BCG injection, mice were perfused and brains were paraffin-embedded for VEGF expression analysis. We observed VEGF immunohistochemical expression in TBM and granulomas in non-vaccinated mice. The DNA-hsp65 treatment blocked the expression of VEGF in mice TBM. Therefore, our murine model indicated the VEGF participation in the physiopathology of CNS-TB and the potential prevention of the DNA-hsp65 in the disease progression.

Copyright © 2013 Elsevier Ltd. All rights reserved.

PMID: 23491717



Angiogenesis is an important phenomenon during normal development and also present in several pathologies. For example, it is essential for tumor growth, metastasis and infectious process, such as CNS-TB. Angiogenesis is a process tightly regulated by the actions of angiogenic mediators, as the endothelial growth factor (VEGF), released by inflammatory cells. VEGF is a heparin-binding dimeric polypeptide growth factor originally purified on the basis of its vascular permeability-enhancing activity and shown to be a potent mitogen for endothelial cells. It induces breakdown of the BBB and subsequent increase in the vascular permeability. VEGF plays a pivotal role in angiogenesis and acts mainly through two tyrosine kinase receptors, flt-1 (VEGFR-1) and flk-1 (VEGFR-2). Increased levels of VEGF are detected in serum of patients with active pulmonary TB and tuberculous meningitis, which also presents VEGF increase in the cerebrospinal fluid (CSF). Therefore, the increase in vascular permeability and angiogenesis recognized to the VEGF production might have essential role in the progression and/or dissemination of the TB disease.


The importance of this study is two-fold:

First, the objective of this study was to extent the characterization of our previously described murine model of central nervous system TB (CNS-TB). TB was neglect for decades. However, the immense public health impact of TB is now widely recognized. The most severe form of TB is that involving the CNS. And the reactivation of M. tuberculosis infection tends to be an early complication and HIV-infected patients with TB, which are at increased risk of meningitis. Furthermore, there is a pressing need for new and better vaccines to fight TB. In this study we tested the experimental effectiveness of a DNA vaccination, composed by a single mycobacterial antigen (DNA-hsp65).

Second, the goal of this study was to describe VEGF participation in the CNS-TB, and its modulation by DNA-hsp65 vaccine. The analysis of the VEGF expression might help to establish a relationship in the VEGF increment with the TB progression and to evaluate the impact of the DNA-hsp65 treatment on the biochemical and morphological compromising in the CNS-TB development. Although only further research will determine the exact nature of VEGF role in CNS-TB, our data indicate that VEGF plays a critical role in TB progression in CNS.


Figure Captions

Fabiola Zucchi-fig1Figure 1. DNA-hsp65 vaccine modulates angiogenic factor VEGF in this experimental model of CNS-TB. Time-course of the procedures: male C57BL/6 mice received 4 doses of DNA-hsp65 vaccine (intramuscularly: femural quadriceps) at 2-week intervals; Mycobacterium bovis bacillus Calmette-Guerin was thus injected intra-cerebellum for induction of meningitis and granulomas; after four weeks animals were sacrificed for immunohistochemical characterization of VEGF expression.

 Fabiola Zucchi-fig2Figure 2. Immuno-histochemical VEGF expression in the experimental model of CNS-TB, in mice with or without previous DNA-hsp65 immunization (vaccine against TB). A. Tuberculous lesion intra-parenchyma (arrow), VEGF stained. VEGF immuno-reactive meningitis (arrow head). Magnitude: 20X. B. Stained blood vessel (arrow) near lesion showed in A. C. Lymphocytic meningitis without VEGF stain between cerebellar lobes (arrow) in DNA-hsp65 vaccinated mice. 20X. D. Meningitis (arrow – idem C; 60X). Abbreviation: MOL, molecular layer; PCL, Purkinje cell layer; GL, granular layer.



  1. Zucchi FCR, Pelegrini-da-Silva A, Neder L, Silva CL, Tsanaclis AM, Takayanagui OM (2012) The contribution of a murine CNS-TB model for the understanding of the host-pathogen interactions in the formation of granulomas. J Neurosci Methods, 206(1):88-93.
  2. Zucchi FCR, Tsanaclis AM, Moura-Dias Jr Q, Silva CL, Pelegrini-da-Silva A, Neder L, Takayanagui OM (2013) Modulation of angiogenic factor VEGF by DNA-hsp65 vaccination in a murine CNS tuberculosis model. Tuberculosis, 93:373-380.



The authors thank Dr. Luciana Leite (Butantã Institute – São Paulo, Brazil) for kindly donating the BCG samples, Dr. William Alves Prado (FMRP-USP), and Dr. Guillaume Sébire (Université de Sherbrooke) for providing the laboratory infrastructure, as well as Vani Alves, Ana Anselmi, Paulo Castania, Maria Paula Scandar, Renata Caldo (FMRP-USP), Jocelyne Lapointe and Annie Larouche (Université de Sherbrooke) for their technical expertise. This study received financial support in the form of PhD and PDEE (Programa de Doutorado no País com Estágio no Exterior) fellowship for F.C.R.Z. from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Coordination of the Advancement of Higher Education). Recently Dr. Fabiola Zucchi received a grant from the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, National Council for Scientific and Technological Development) Chamada Universal 14/2013 for follow up investigations.



Fabiola Cristina Ribeiro Zucchi – PhD

Visiting Assistant Professor

State University of Mato Grosso, Faculty of Health Sciences, Department of Medicine (F.C.R. Zucchi)

Fabiola Zucchi-fig3

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