Profile of time-dependent VEGF upregulation in human pulmonary endothelial cells, HPMEC-ST1.6R infected with DENV-1, -2, -3, and -4 viruses
© Azizan et al; licensee BioMed Central Ltd. 2009
Received: 19 March 2009
Accepted: 06 May 2009
Published: 06 May 2009
In this study, the upregulated expression level of vascular endothelial growth factor (VEGF) in a pulmonary endothelial cell line (HPMEC-ST1.6R) infected with dengue virus serotypes 1, 2, 3, and 4 (DENV-1, -2, -3 and -4), was investigated. This cell line exhibits the major constitutive and inducible endothelial cell characteristics, as well as angiogenic response. Infection by all four DENV serotypes was confirmed by an observed cytopathic effect (CPE), as well as RT-PCR (reverse-transcription polymerase chain reaction) assays. As we had previously reported, the DENV-infected HPMEC-ST1.6R cells exhibited an elongated cytoplasmic morphology, possibly representing a response to VEGF and activation of angiogenesis. In this study, increase in VEGF expression level at designated time points of 0, 8, 24, 96 and 192 hours post-infection was investigated, using a microbead-based Bio-Plex immunoassay. Increased level of VEGF expression in infected-HPMEC-ST1.6R was detected at 8 hours post-infection. Interestingly, VEGF expression level began to decrease up to 96 hours post-infection, after which an upsurge of increased VEGF expression was detected at 192 hours post-infection. This profile of VEGF upregulated expression pattern associated with DENV infection appeared to be consistent among all four DENV-serotypes, and was not observed in mock-infected cells. In this study, the expression level of VEGF, a well-established vascular permeabilizing agent was shown to be elevated in a time-dependent manner, and exhibited a unique dual-response profile, in a DENV-infected endothelial cell. The experimental observation described here provided additional insights into potential mechanism for VEGF-mediated vascular leakage associated with DENV, and support the idea that there are potential applications of anti-VEGF therapeutic interventions for prevention of severe DENV infections.
Dengue fever (DF) and dengue hemorrhagic fever (DHF) are caused by one of four closely related, but antigenically distinct, dengue virus serotypes 1, 2, 3 or 4 (DENV-1, DENV-2, DENV-3, and DENV-4) [1, 2]. Several clinical manifestations including plasma leakage, thrombocytopenia and hemorrhage, distinguish DHF from DF, which is a milder infection [2–4]. Severe DHF, also known as dengue shock syndrome (DSS), can occur when fluid leakage into interstitial tissue spaces leads to hypovolemic shock. Plasma levels of various cytokines such as TNF-α, IFN-γ and IL-8, were found to be significantly higher in DHF patients, when compared to DF patients . DENV infection of target cells in-vitro, induces increased expression level of these cytokines and other growth factors such as VEGF. Previous studies have inferred that endothelial cell damage may also be mediated through effects of induced VEGF expression in DENV-infected cells [5–8]. These perturbations are thought to at least be partly responsible for some of the clinical manifestations of hemorrhage and capillary leakage associated with DHF.
VEGF levels (pg/ml) ± SD in cell culture conditioned medium of HPMEC-ST1.6R endothelial cells that were infected with DENV-1, -2. -3 and -4, or mock-infected [VM(-)] with vero cells culture medium.
VM (-) Mock
0 ± 1.4
0 ± 5.7
0 ± 0.4
0 ± 1.4
0 ± 0
1,244.0 ± 1.4
14,997.5 ± 50.2
14,223.8 ± 460.3
21,275.6 ± 550.5
9,094.5 ± 220.6
600.1 ± 59.4
10,167.1 ± 384.3
11,039.2 ± 969.4
15,529.7 ± 238.3
11,782.0 ± 53.7
876.0 ± 28.6
6,150.6 ± 730.1
10,349.4 ± 189.9
13,541.0 ± 523.6
4,610.1 ± 207.9
3,247.7 ± 398.5
25,948.5 ± 432.0
29,889.6 ± 828.4
41,560.4 ± 131.2
20,281.7 ± 706.1
P-values for VEGF increased production levels comparing between DENV-infected and mock-infected HPMEC-ST1.6R endothelial cells.
Many studies have shown that vascular hyper-permeability which can lead to vascular leakage, can occur in response to a single, brief exposure in the endothelium to VEGF or other vascular permeabilizing agents . VEGF, which is the most well-characterized pro-angiogenic growth factor, is involved not only in promoting angiogenesis which produces new blood vessels, but also in stimulating endothelial cell proliferation, migration, differentiation, tube formation, increased vascular permeability and maintaining vascular permeability . Endothelial cells infected by another viral agent, Hepatitis C virus , and epithelial cells infected by human rhinovirus  were also shown to induce increased expression level of VEGF, implicating their roles in mediating immunopathologies associated with these specific viral infections. Hantaviruses which cause two lethal vascular permeability-based diseases; hemorrhagic fever with renal syndromes and hantavirus pulmonary syndromes  was reported to specifically enhance VEGF-directed permeabilizing responses in infected endothelial cells. This particular study implicated a direct role for VEGF in mediating vascular leakage and hemorrhagic diseases in these Hantaviruses-associated diseases and other vascular leakage syndromes.
Severity of plasma leakage in DENV patients was found to correlate with increased plasma levels of VEGF , but inversely correlated to soluble vascular endothelial growth factor receptor 2 (sVEGFR2) . Interestingly, plasma viral load correlated well with a decline of VEGFR2, which is believed to bind to VEGF, controlling its availability and interfering with its cellular function. One proposed implication from this study was that VEGF participates in regulating vascular permeability that leads to plasma leakage seen in DHF patients, and that its activity and availability is controlled by a soluble form of its receptor, sVEGFR2. A related study which was cited earlier  showed that hantavirus-directed permeability in infected endothelial cell could be inhibited by antibodies to VEGFR2, which implicates its therapeutic potential in the treatment of vascular leakage and hemorrhagic diseases. We have shown in our study that the expression level of VEGF was elevated in a time-dependent manner, and exhibited a unique dual-response profile in endothelial cells infected by all four serotypes of DENV viruses. The experimental observation described here could provide insights into potential mechanism for VEGF-mediated vascular leakage associated with DENV. Findings from this and other related studies could provide impetus to further establish use of anti-VEGFR2 and other potential anti-VEGF agents [19–21] as therapeutic interventions for treatment and prevention of vascular leakage associated with DHF.
We thank Dr. Vera Krump-Konvalinkova and Dr. C.J. Kirkpatrick at The Institute of Pathology, Johannes Gutenberg University, Mainz, Germany for the kind gift of HPMEC-ST1.6R cell line used in this study. We thank Dr. Dennis Kyle for his permission to use the MXPro3000P (Stratagene) real time PCR instrument for this study. Funding was provided for by the Southeastern Center for Emerging Biologic Threats (SECEBT).
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