Systemic and Cerebral Vascular Endothelial Growth Factor Levels Increase in Murine Cerebral Malaria along with Increased Calpain and Caspase Activity and Can be Reduced by Erythropoietin Treatment

Abstract

The pathogenesis of cerebral malaria (CM) includes compromised microvascular perfusion, increased inflammation, cytoadhesion, and endothelial activation. These events cause blood-brain barrier disruption and neuropathology and associations with the vascular endothelial growth factor (VEGF) signaling pathway have been shown. We studied this pathway in mice infected with Plasmodium berghei ANKA causing murine CM with or without the use of erythropoietin (EPO) as adjunct therapy. ELISA and western blotting was used for quantification of VEGF and relevant proteins in brain and plasma. CM increased levels of VEGF in brain and plasma and decreased plasma levels of soluble VEGF receptor 2. EPO treatment normalized VEGF receptor 2 levels and reduced brain VEGF levels. Hypoxia-inducible factor (HIF)-1α was significantly upregulated whereas cerebral HIF-2α and EPO levels remained unchanged. Furthermore, we noticed increased caspase-3 and calpain activity in terminally ill mice, as measured by protease-specific cleavage of α-spectrin and p35. In conclusion, we detected increased cerebral and systemic VEGF as well as HIF-1α, which in the brain were reduced to normal in EPO-treated mice. Also caspase and calpain activity was reduced markedly in EPO-treated mice.

Keywords: VEGF; brain; cerebral malaria; erythropoietin; hypoxia; inflammation; neuropathology.

Figure 1

Progression of body temperature and parasitemia during the course of infection. (A) Body temperature remained stable until day 6 p.i. and increased slightly in infected mice. InfSal mice displayed clinical signs of CM at day 8 p.i. and had significantly lowered body temperature (p < 0.005). (B) Parasitemia rose gradually in both groups but did not increase as fast in InfEPO mice from day 7 p.i and onward (p < 0.05). Data are represented as mean values and error bars display standard deviation. Significant deviations from uninfected, saline-treated mice are denoted with an asterisk.

Figure 2

Cerebral expression of HIF-1α, VEGF, HIF-2α, and EPO. (A) HIF-1α levels were significantly increased in InfSal mice compared with UninfSal (p = 0.04). The other groups were statistically indistinguishable. (B) VEGF was analyzed with ELISA, showing a significant increase in InfSal mice compared with uninfected mice (p = 0.02). InfEPO was increased though not statistically significantly. (C) HIF-2α expression was similar in all four groups. (D) Also, EPO was expressed in the same level in all four groups. Bar charts (A) show mean values and standard deviation. Box plots (B–D) show median values and interquartile ranges. Whiskers show Tukey hinges; open circles are outliers. Asterisks denote significant deviations from uninfected, saline-treated mice.

Figure 3

Calpain and caspase activity in terminal CM. Activity of caspase-3 and calpain was assessed by looking for specific cleavage products. (A) Analyzing the 150 kDa fragment of α-spectrin specific for calpain activity show a significant increase in InfSal mice (p = 0.04) compared with uninfected mice. (B) No significant change was noted when analyzing the 145 kDa fragment, also specific for calpain activity. (C) The 120 kDa band specific for caspase-3 activity was markedly increased in InfSal mice (p < 0.01). (D) p35 is cleaved by calpain activity into a smaller fragment, p25. This fragment was not detectable in UninfEPO mice, but was significantly increased in InfSal mice (p < 0.05). Box plots show median values and interquartile ranges. Whiskers show Tukey hinges; open circles are outliers. Asterisks denote significant deviations from uninfected, saline-treated mice.

Figure 4

Plasma levels of VEGF, soluble Flt-1 and soluble Flk-1. Plasma was extracted from euthanized mice at day 8 p.i. and analyzed by ELISA. (A) VEGF levels increased significantly due to infection (p < 0.05). Both infection groups had significantly increased plasma levels of VEGF (p = 0.04 saline-treated, p = 0.03 EPO-treated). (B) Soluble Flt-11 was largely unaffected by infection and treatment though significant changes were noted (p = 0.02). No groups deviated from UninfSal mice (p > 0.14). (C) Soluble Flk-1 was significantly affected by both EPO treatment and infection. Soluble Flk-1 levels were significantly decreased due to CM (p < 0.001). On the contrary, EPO treatment led to an increased level of this receptor in both infected (p = 0.03) and uninfected mice (p = 0.02) compared with the corresponding saline-treated control groups. Yet, infection strongly decreased sFlk-1 levels in EPO-treated mice (p < 0.001) compared with UninfEPO mice. (D) When taking the ratio of sFlt-1 to sFlk-1 only InfSal was significantly different from uninfected mice (p<0.002). Box plots (A,B,D) show median values and interquartile ranges. Whiskers show Tukey hinges; open circles are outliers. Strip chart (C) show the mean value as a cross and whiskers represent standard deviation. Each dot represents the plasma level in one mouse. Asterisks denote significant deviations from uninfected, saline-treated mice.

Figure 5

Plasma angiopoietin-1 levels are affected by infection and EPO treatment. In terminal CM, plasma angiopoietin-1 levels were significantly decreased (p = 0.004). The, InfEPO group also had lower levels of this cytokine but was statistically similar to uninfected mice (p = 0.06). Box plots show median values and interquartile ranges. Whiskers show Tukey hinges; open circles are outliers. Asterisks denote significant deviations from uninfected, saline-treated mice.