Profile of time-dependent VEGF upregulation in human pulmonary endothelial cells, HPMEC-ST1.6R infected with DENV-1, -2, -3, and -4 viruses

Abstract

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.

Figure 1

RT-PCR analysis of DENV-1, -2, -3 and 4 infected HPMEC-ST1.6R cells. Cell culture media of HPMEC-ST1.6R cells infected with DENV-1, -2, -3 and -4 were collected and used to obtain total RNA. A real-time RT-PCR analysis of HPMECST1.6R cells infected with DENV-1, -2, -3 and -4 were subsequently performed using the extracted RNA for quantitative analysis, using the MXPro3000P [Stratagene]. A SYBR Green 1 kit [Stratagene] and the DN-F/DN-R primer set [10] were utilized for amplifications, following instructions specified by the manufacturer. Following amplification, melting curve analysis was performed by raising the incubation temperature from 62°C to 95°C to verify correct amplification product by its specific melting temperature. DENV specific products derived from Vero or HPMEC cells infected with DENV-1 displayed a T_m of 80.7°C, while DENV-2, -3 and -4 displayed specific products with T_m values of 81.8°C, 80.1°C and 81.2°C, respectively.

Figure 2

VEGF Production in HPMEC-ST1.6R Cells infected with DENV-1, -2, -3, and -4. Monolayers of HPMEC-ST1.6R cells were infected with either DENV-1, -2, -3 or DENV-4 viruses as previously described [6], and incubated at 37°C in 5% CO₂ for 6 days. The cell media of DENV-infected cells and mock-infected cells were collected at 0, 8, 24, 96 and 192 hours post-infection, and analyzed for VEGF production [actual values listed in Table 1]. Mean VEGF levels were determined using a VEGF analyte detection kit and a BioPlex suspension array analyzer from BioRad. Significant increases (p ≤ 0.05) in cytokines between virus-infected and mock-infected cell cultures are given in Table 2. [Bars equal standard deviation from the mean of triplicates].