Induction of Angiogenesis by Malarial Infection through Hypoxia Dependent Manner

Abstract

Malarial infection induces tissue hypoxia in the host through destruction of red blood cells. Tissue hypoxia in malarial infection may increase the activity of HIF1α through an intracellular oxygen-sensing pathway. Activation of HIF1α may also induce vascular endothelial growth factor (VEGF) to trigger angiogenesis. To investigate whether malarial infection actually generates hypoxia-induced angiogenesis, we analyzed severity of hypoxia, the expression of hypoxia-related angiogenic factors, and numbers of blood vessels in various tissues infected with Plasmodium berghei. Infection in mice was performed by intraperitoneal injection of 2×106 parasitized red blood cells. After infection, we studied parasitemia and survival. We analyzed hypoxia, numbers of blood vessels, and expression of hypoxia-related angiogenic factors including VEGF and HIF1α. We used Western blot, immunofluorescence, and immunohistochemistry to analyze various tissues from Plasmodium berghei-infected mice. In malaria-infected mice, parasitemia was increased over the duration of infection and directly associated with mortality rate. Expression of VEGF and HIF1α increased with the parasitemia in various tissues. Additionally, numbers of blood vessels significantly increased in each tissue type of the malaria-infected group compared to the uninfected control group. These results suggest that malarial infection in mice activates hypoxia-induced angiogenesis by stimulation of HIF1α and VEGF in various tissues.

Keywords: HIF1α; Plasmodium berghei; VEGF; angiogenesis; hypoxia; malaria.

Fig. 1

Parasitemia and survival of infected mice. (A) Parasitemia, and (B) survival of C57BL/6 mice infected with 10⁶ parasitized erythrocytes by P. berghei.

Fig. 2

Expression of HIF1α and VEGF in various malaria-infected tissues. (A) Western blot analysis, and quantified expression of (B) HIF1α, and (C) VEGF. Expression was analyzed by western blotting for tissues from control (not infected), and infected mice (10%, and 30% parasitemia). Four or five different mice per each time-point were sacrificed and total lysates were analyzed. Quantification of each protein expression was calculated by band densities by image analysis software (ImageJ) and normalized using the average value for GAPDH. Expression level shows mean value and 95% confidence intervals (CIs). The 2-tailed Student’s t-test was performed to evaluate statistical significance among groups. *P<0.05, **P<0.01.

Fig. 3

Immunofluorescence imaging of a hypoxia marker and HIF1α in malaria-infected tissues (brain, heart, kidney, liver, lung, and muscle) with parasitemia levels of 0%, 10%, and 30%. Original magnification was ×400 for immunofluorescence. Hypoxic condition and the expression of HIF1α was increased in various infected tissues and HIF1α protein was partially colocalized with hypoxic area.

Fig. 4

Immunofluorescence imaging of VEGF in malaria-infected tissues (brain, heart, kidney, liver, lung, and muscle) with parasitemia levels of 0%, 10%, and 30%. Original magnification was ×400 for immunofluorescence. The expression of HIF1α was increased in various infected tissues.

Fig. 5

Angiogenic stimulation by malarial infection. Immunohistochemical staining of blood vessels in tissues with parasitemia levels of 0%, 10%, and 30% with blood endothelial cell marker CD31 antibody. Arrows indicate the blood vessels stained with CD31 antibody.

Fig. 6

Angiogenic stimulation by malarial infection. Number of blood vessels in tissues with parasitemia levels of 0%, 10%, and 30%. Number of blood vessels was counted at 20 fields (×200) and calculated mean value and 95% confidence intervals (CIs). The 2-tailed Student’s t-test was performed to evaluate statistical significance among groups. *P<0.05, **P<0.01, ***P<0.001.