Endothelial progenitor cells and the pathogenesis of cerebral malaria

描述（由申请人提供）：疟疾是全世界死亡的主要原因。大多数死亡是由于感染恶性疟原虫（P. falciparum）。脑型疟疾（CM）是这些患者死亡的主要原因。尽管其毒力，CM的病理生理基础是知之甚少。最近的研究表明，微血管损伤是与血脑屏障功能障碍/破坏相关的因素。内皮细胞损伤是由几个因素引起的，包括：受感染的红细胞的隔离，缺氧损伤和“细胞因子风暴”。受损微血管的修复可以通过局部内皮细胞的增殖或骨髓来源的循环内皮祖细胞（cEPCs）的募集来发生。cEPCs的低水平和功能障碍与微血管损伤相关疾病（包括心血管疾病、糖尿病和中风）的症状和不良事件的发生相关。循环EPC个体发生和从骨髓的迁移由趋化因子/蛋白酶如基质衍生生长因子1（SDF-1）和基质金属蛋白-9（MMP-9）介导。我们假设恶性疟原虫感染导致微血管损伤和修复之间的不平衡。当cEPCs减少并且受损的内皮细胞不能被替换时，发生脑型疟疾。R21支持的研究支持这一假设。加纳CM儿童中cEPC的平均百分比显著低于无并发症疟疾（UM）、无症状寄生虫血症（AP）或健康对照（HC）儿童（p<0.0001）。此外，与AP或HC儿童相比，CM和UM儿童的平均血浆SDF-1水平显著升高（p<0.001）。该提案的具体目标将进一步定义宿主对疟疾微血管损伤的反应，并确定其与CM发展或恢复的关系。将加纳不同疾病严重程度（CM、UM和AP）的恶性疟原虫感染儿童与健康对照组进行比较。目的1）确定宿主对微血管损伤反应的时间进程。将在同一患者中前瞻性测量cEPC、SDF-1和MMP-9的水平。我们预测发生CM的UM患者应具有导致CM发生的cEPC水平降低。在CM恢复的患者中应检测到cEPCs的增加。SDF-1和MMP-9水平应在发生CM的患者中升高，并随恢复而下降。目的2）探讨循环内皮祖细胞在疟疾感染中的作用。将进行Transwell迁移试验和血液生长内皮细胞培养以及DiLDL代谢标记。我们预测CM患者的cEPCs将具有功能异常。目的3）评估用于测定cEPC水平的qRT-PCR方法。定量RT-PCR可能是一种用于流行地区确定cEPC水平以及评估功能的简便工具。将通过与流式细胞术分析和功能测定进行比较来评估qRT-PCR的效用。

DESCRIPTION (provided by applicant): Malaria is a major cause of mortality worldwide. The majority of deaths are due to infections with Plasmodium falciparum (P. falciparum). Cerebral malaria (CM) is a major cause of death in these patients. Despite its virulence, the pathophysiologic basis of CM is poorly understood. Recent investigations suggest that microvascular damage is a factor with associated dysfunction/ dysruption of the blood brain barrier. Endothelial cell damage is caused by several factors including: the sequestration of infected red blood cells, hypoxic damage and "cytokine storm". Repair of damaged microvasculature may occur either by the proliferation of local endothelial cells or the recruitment of bone-marrow derived circulating endothelial progenitor cells (cEPCs). Low levels and dysfunction of cEPCs have been associated with the development of symptoms and adverse events in diseases associated with microvascular damage including cardiovascular disease, diabetes and stroke. Circulating EPC ontogeny and migration from the bone marrow is mediated by chemokines/proteases such as the stromal derived growth factor 1 (SDF-1) and the matrix metalloprotein-9 (MMP-9). We hypothesize that P. falciparum infection results in an imbalance between microvascular damage and repair. Cerebral malaria occurs when cEPCs are diminished and damaged endothelial cells cannot be replaced. A R21 supported study supports this hypothesis. The mean percentage of cEPCs are significantly lower in Ghanaian children with CM as compared to those with uncomplicated malaria (UM), asymptomatic parasitemia (AP) or healthy controls (HC) (p<0.0001). In addition, mean plasma levels of SDF-1 are significantly increased in children with CM and UM as compared to those with AP or HC (p<0.001). The Specific Aims of this proposal will further define the host response to microvascular damage in malaria and determine its association with the development of or recovery from CM. P. falciparum-infected children in Ghana with different degrees of disease severity (CM, UM and AP) will be compared with healthy controls. Aim 1) To determine the time course of the host response to microvascular damage. Levels of cEPCs, SDF-1 and MMP-9 will be measured prospectively in the same patient. We predict that patients with UM who develop CM should have decreasing cEPC levels leading to the development of CM. Increases in cEPCs should be detected in patients who recover from CM. SDF-1 and MMP-9 levels should be elevated in patients who develop CM and decline with recovery. Aim 2) To assess the function of circulating EPCs in malaria infections. Transwell migration assays and blood outgrowth endothelial cell culture coupled with DiLDL metabolic labeling will be performed. We predict that the cEPCs of patients with CM will have functional abnormalities. Aim 3) To assess qRT-PCR assays for the determination of cEPC levels. Quantitative RT-PCR is potentially a facile tool for use in endemic areas for determining cEPC levels as well as assessing function. The utility of qRT-PCR will be assessed in comparison to flow cytometry analysis and functional assays.