Manipulation of Host Angiogenesis as a Therapeutic Strategy against Invasive Pulm

描述（由申请人提供）：侵袭性曲霉病（IA）是免疫功能低下患者呼吸道和播散性感染的重要原因，也是造血干细胞移植受者感染性肺炎死亡的最常见原因。尽管有新型抗真菌药物可用，但 IA 的死亡率仍然高得令人无法接受。抗真菌药物对 IA 的疗效不佳可能与曲霉菌侵入肺血管的倾向有关，导致血管内血栓形成、组织缺血和梗死。这种血管病变隔离受感染的组织，从而限制抗真菌药物向感染部位的输送。此外，IA 与编码血管生成重要介质的基因表达下调相关，例如血管内皮生长因子 (VEGF) 和碱性成纤维细胞生长因子 (bFGF)，表明血管对 IA 的反应受到抑制。我们假设促血管生成生长因子与抗真菌药物一起治疗性给药将通过增加抗真菌药物的组织浓度来提高实验 IA 中的存活率。在目标 1 中，我们建议评估单独使用促血管生成生长因子或与抗真菌药物两性霉素 B (AMB) 联合治疗 IA 的可行性。为此，我们将使用环磷酰胺诱导中性粒细胞减少症后的两种 IA 小鼠模型：（1）急性肺部模型，其中通过鼻内滴注浓缩孢子悬浮液建立感染； (2)亚急性模型，通过皮下注射孢子悬浮液诱导肌肉皮肤感染。在每个模型中，将评估以下治疗组：VEGF、bFGF、VEGF加bFGF、AMB、AMB加VEGF、AMB加bFGF以及AMB加VEGF和bFGF。另外两组小鼠将接受舒尼替尼（一种血管生成抑制剂）单独或与 AMB 联合治疗。将评估和比较治疗组之间的三种类型的终点： (1) 7 天的存活率（仅肺模型）； (2) 组织真菌负荷，通过定量聚合酶链反应（qPCR）测量； (3)感染部位的血管生成，使用先前描述的基质胶测定在肌皮肤模型中体内评估，并使用微血管密度在肺组织切片中评估。在目标 2 中，我们将确定 VEGF 和 bFGF 治疗对 AMB 组织浓度的影响。使用高效液相色谱法测量肺组织和基质胶塞中的 AMB 浓度，并在单独接受 AMB 的小鼠组和接受 AMB 加 VEGF 和/或 bFGF 的小鼠组之间进行比较。公共卫生相关性：侵袭性曲霉病是癌症和免疫系统减弱患者患病和死亡的主要原因。尽管有新的抗真菌药物和改善的支持治疗，这些感染的死亡率仍然高得令人无法接受。我们假设，侵袭性曲霉菌病过程中的血管闭塞限制了抗真菌药物渗透到感染组织中，而使用促进新血管形成的生长因子进行治疗可能会改善这种严重真菌感染的结果。

DESCRIPTION (provided by applicant): Invasive aspergillosis (IA) is an important cause of respiratory and disseminated infection in immunocompromised patients, and the most common cause of infectious pneumonic mortality in recipients of hematopoietic stem cell transplants. Mortality from IA remains unacceptably high despite the availability of novel antifungal agents. The poor efficacy of antifungal drugs against IA may be linked to the propensity of Aspergillus species to invade pulmonary blood vessels, causing intravascular thrombosis, tissue ischemia and infarction. This vasculopathy sequesters infected tissue, thereby limiting the delivery of antifungal agents to the site of infection. Furthermore, IA is associated with down-regulation of the expression of genes encoding for important mediators of angiogenesis, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), suggesting that the vascular response to IA is suppressed. We hypothesize that therapeutic administration of pro-angiogenic growth factors together with antifungal drugs will increase the survival rate in experimental IA by increasing the tissue concentrations of the antifungal drugs. In aim 1, we propose to assess the feasibility of treating IA with pro-angiogenic growth factors, alone or in combination with the antifungal drug amphotericin B (AMB). To that end, we will use two murine models of IA following induction of neutropenia with cyclophosphamide: (1) An acute pulmonary model, in which infection is established by intranasal instillation of a concentrated spore suspension; (2) A subacute model, in which myocutaneous infection is induced by subcutaneous injection of spore suspension. In each model, the following treatment groups will be assessed: VEGF, bFGF, VEGF plus bFGF, AMB, AMB plus VEGF, AMB plus bFGF, and AMB plus VEGF and bFGF. Two additional groups of mice will receive treatment with sunitinib, an inhibitor of angiogenesis, alone or in combination with AMB. Three types of endpoints will be assessed and compared among treatment groups: (1) Survival over a period of 7 days (pulmonary model only); (2) Tissue fungal burden, measured by quantitative polymerase chain reaction (qPCR); and (3) Angiogenesis at the site of infection, assessed in vivo in the myocutaneous model using the previously described matrigel assay, and in pulmonary tissue sections using microvessel density. In aim 2, we will determine the effect of treatment with VEGF and bFGF on the tissue concentration of AMB. AMB concentrations will be measured in pulmonary tissue and in matrigel plugs using high performance liquid chromatography, and compared between groups of mice receiving AMB alone and groups receiving AMB plus VEGF and/or bFGF. PUBLIC HEALTH RELEVANCE: Invasive aspergillosis is a major cause of sickness and death in patients with cancer and a weakened immune system. Despite the availability of new antifungal drugs and improved supportive care, mortality from these infections remains unacceptably high. We hypothesize that the occlusion of blood vessels in the course of invasive aspergillosis limits the penetration of antifungal drugs into infected tissue, and that treatment with growth factors that enhance the formation of new vessels might improve the outcome of this severe fungal infection.