Plasma concentrations of Ang-2 and the Ang-2/Ang-1 ratio were confirmed as independent predictors of death in Vietnamese adults with severe falciparum malaria, as seen in all other adequately powered severe malaria series studied to date. These include series of adult severe malaria from Indonesia , adult CM from India , and paediatric CM series from Uganda [26, 27] and Malawi [28, 29]. Patterns of immunostaining of Ang-1, Ang-2 and Tie-2 in malaria cases were altered in fatal severe malaria compared to fatal non-malaria cases, especially in neurons and astroglial cells. However, there were no specific patterns of Ang-1, Ang-2 and Tie-2 expression, either of neuronal, astroglial or vascular, that differentiated CM from non-cerebral severe malaria deaths. Activation of the Ang-Tie-2 pathway in severe malaria was related to acidosis, the number of severity criteria and outcome, but was not a specific event in the brain during CM.
Previous pathological studies of CM have demonstrated the presence of EC activation in the cerebral microvasculature , which occurs as part of a wider systemic endothelial activation in severe malaria, and which is not specific to cases that develop coma . The pathophysiology of endothelial activation in CM has been hypothesized to occur in response to both systemic factors (such as the release of cytokines or a soluble malaria toxin) and local vasoactive substances such as VEGF or WPB products such as Ang-2. The expression of mediators, which could influence endothelial reactivity in the brain in CM has been previously examined, including inducible nitric oxide synthase , the hypoxic inducible protein HIF-1a and activation of the VEGF signalling pathway . Therefore this methodology was extended to examine the expression of the angiopoietins and their receptor Tie-2 in the brain in CM and NCM cases, to determine the extent to which they were specific to CM and whether this pathway showed a direct link to the clinical features of coma before death.
The lack of significant differences in the patterns or extent of staining of any of these constituents between CM and NCM cases is consistent with recent data using systemic measures of activation of the Ang-2-Tie2 pathway, where elevated plasma levels of Ang-2 were no higher in CM than in NCM . Taken together, these results do not support elevated Ang-2 as a predictive biomarker for cerebral malaria, as previously proposed [25, 26]. Indeed in the current study, circulating Ang-2 was elevated less in those with CM as the only manifestation than in those with other severe malaria criteria.
The low levels of Ang-2 expression in brain ECs in fatal CM and NCM, despite elevated plasma concentrations of Ang-2, was unexpected. It is possible that exocytosis of cerebral endothelial WPBs at the time of death decreased immunoreactivity, due to prolonged EC activation, as stored Ang-2 is rapidly released from WPBs on activation . Alternatively, raised Ang-2 levels in serum may reflect release from the extra-cerebral systemic circulation in both CM and NCM.
As well as its independent association with fatal outcome, Ang-2 was associated with number of organ complications, metabolic acidosis, and acute renal failure, as previously shown . In contrast, the other major WPB product increased in severe malaria, vWF, is not associated with either lactate/metabolic acidosis or fatal outcome in severe malaria [39, 43]. Taken together, these findings do still suggest a specific pathogenic role for Ang-2 in the pathways leading to death in severe malaria. Ang-2 competitively binds to Tie-2 receptors blocking the homeostatic effects of Ang-1, increasing endothelial activation and sensitising ECs to further activation and injury . Ang-2 related amplification of endothelial activation, injury and microvascular sequestration may thus play a key role in impairment of microvascular perfusion in severe malaria.
Potential limitations to interpretation of immunohistochemical staining patterns include post mortem artefact, which may alter staining due to diffusion or degradation of proteins, and necessitated the use of control cases. Each death in an autopsy series gives a ‘snapshot’ effect of pathology in an individual case with varying treatment, time to death and clinical complications of disease, which differ between cases. Inferring a single unifying pattern of pathology with temporal sequence from a group of such cases may therefore be difficult. Severe malaria in Vietnamese adults is a multi-organ process with high prevalence of renal, liver, respiratory and metabolic dysfunction as well as anaemia and cerebral malaria. Hence several factors may affect the expression of a particular marker as judged by immunohistochemistry. Despite this, it was reassuring that the data on plasma levels of Ang-1, Ang-2 and Tie-2 were in keeping with previously published series on these mediators in severe and fatal malaria [24–29, 39]. This lends weight to the findings that despite the significant increases in Ang-2 and Ang2/Ang-1 ratio in severe malaria, there was no evidence that this was a process that was specifically upregulated in the brain in CM versus NCM cases.
The analysis of the prognostic value of Ang-2 in severe malaria confirmed, as found in previous studies, that this marker is predictive of fatal outcome, at a level superior to previously studied prognostic biomarkers such as serum lactate. However most studies use a single (admission) value of serum Ang-2 and have not studied the kinetics during severe disease. Lactate levels fall rapidly after treatment but in contrast remain high or rise in patients with fatal outcome despite treatment [44, 45]. Data on the kinetic responses of Ang-2 and Ang-2/Ang-1 ratio following treatment would be valuable in interpreting the specificity of Ang-2 increases in severe malaria.
The effects of Ang-1, Ang-2 and Tie-2 signalling on neurons and glial cells have not been as well studied as endothelium. It was notable that expression of Ang-1, Ang-2 and Tie-2 differed in neurons and glial cells in fatal malaria cases compared to controls. Recent studies indicate that Tie-2 signal transduction might have neuroprotective and mitogenic effects on neuronal cells. Ang-1-Tie-2 signalling promotes neural outgrowth from dorsal root ganglion cells , supports neuronal differentiation in neural progenitor cells , protects against neuronal apoptosis  and the effects of oxygen and glucose starvation , both of which may occur in the brain during the coma of CM. For these reasons, the increased neuronal Ang-1 expression in association with microvascular haemorrhages may reflect an adaptive, neuroprotective response to cerebral disease, where haemorrhages reflect focal damage to BBB function which are associated with perivascular edema formation . In a murine model of angiogenesis during experimental autoimmune encephalomyelitis, temporal changes in neuronal and glial expression of Ang-1 and vascular expression of Tie-2 in the spinal cord was seen in concert with BBB permeability changes . Ang-1 effects on neurons are not exclusively limited to Tie-2-receptor dependence, as Ang-1 can induce neurite outgrowth via the β1-integrin receptor on neurons . Other studies have also found a positive effect of Ang-2 on neurogenesis and migration of neuroblasts . It is therefore possible that increased expression of both Ang-1 and Ang-2 on neuronal cells are neuroprotective responses in severe malaria whether or not this involves coma.
Despite the finding that Ang-2 is associated with poor outcome, the immunohistochemical staining and ELISA data provided no evidence for direct involvement of this pathway in the genesis of coma by increases in CM versus NCM patients. The strong correlation between plasma Ang-2 levels and Ang2/Ang1 ratio with metabolic acidosis and increasing number of clinical severity criteria suggests an association with widespread endothelial activation in severe disease, even if this does not have a specific effect on BBB function in the genesis of coma. However the Ang-2-Tie-2 pathway could be studied using in vitro models of BBB function in the presence of sequestered, malaria-infected erythrocytes to investigate this further. Given the association of plasma Ang-2 with acute renal failure in this and other series, and the relationship with metabolic acidosis in the multivariate analysis, further studies should examine the role of the Ang-Tie-2 pathway in regulating blood flow and microvascular pathology in the kidney in adult severe malaria patients.