Endothelial progenitor cells and the pathogenesis of cerebral malaria

描述(申请人提供)：疟疾是世界范围内死亡的主要原因。大多数死亡是由于感染恶性疟原虫(恶性疟原虫)。脑型疟疾(CM)是这些患者死亡的主要原因。尽管其毒力很强，但对CM的病理生理学基础了解甚少。最近的研究表明，微血管损伤是与血脑屏障功能障碍/障碍相关的一个因素。内皮细胞损伤是由多种因素引起的，包括：感染红细胞的隔离、缺氧性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(CEPC)的募集来实现。在心血管疾病、糖尿病和中风等与微血管损害相关的疾病中，cEPC水平低和功能障碍与症状和不良事件的发生有关。基质衍生生长因子-1(SDF-1)和基质金属蛋白-9(MMP-9)等趋化因子/蛋白酶介导了循环内皮祖细胞的个体发生和迁移。我们假设恶性疟原虫感染导致微血管损伤和修复之间的失衡。当cEPC减少，受损的内皮细胞无法替代时，就会发生脑型疟疾。一项由R21支持的研究支持了这一假设。与无并发症疟疾(UM)、无症状寄生虫血症(AP)或健康对照(HC)儿童相比，患有CM的加纳儿童的cEPC平均百分比显著降低(p&lt；0.0001)。此外，与AP或HC患儿相比，CM和UM患儿血浆SDF-1的平均水平显著升高(p&lt；0.001)。这项提案的具体目标将进一步确定宿主对疟疾微血管损伤的反应，并确定其与CM的发展或康复的关联。加纳感染恶性疟原虫的儿童具有不同程度的疾病严重程度(CM、UM和AP)，将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程。CEPC、SDF-1和MMP-9的水平将在同一患者中进行前瞻性测量。我们预测发生CM的UM患者的cEPC水平降低会导致CM的发生。在从CM恢复的患者中应检测到cEPC的增加。在发生CM的患者中，SDF-1和MMP-9水平应升高，并随着病情的好转而下降。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迁移分析和血液生长内皮细胞培养，并结合DiLDL代谢标记。我们预测，CM患者的cEPC将出现功能异常。目的3)评价定量逆转录聚合酶链式反应(qRT-PCR)检测cEPC水平的可行性。定量RT-PCR可能是一种在流行地区用于测定cEPC水平和评估功能的简便工具。定量逆转录聚合酶链式反应的实用性将通过与流式细胞术和功能分析的比较来评估。

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.