Manipulation of Host Angiogenesis as a Therapeutic Strategy against Invasive Pulm

描述(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。尽管有新的抗真菌药物可用，但IA的死亡率仍然高得令人无法接受。抗真菌药物对IA的疗效较差可能与曲霉菌入侵肺血管的倾向有关，导致血管内血栓形成、组织缺血和梗塞。这种血管病变隔离了感染组织，从而限制了抗真菌药物对感染部位的输送。此外，IA与血管生成的重要介质，如血管内皮生长因子(VEGF)和碱性成纤维细胞生长因子(BFGF)等编码基因的表达下调有关，表明血管对IA的反应受到抑制。我们推测，促血管生成生长因子联合抗真菌药物的治疗性应用将通过增加抗真菌药物的组织浓度来提高实验性IA的存活率。在目标1中，我们建议评估促血管生成生长因子单独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后的两种IA小鼠模型：(1)急性肺模型，其中通过鼻内滴注浓缩孢子悬液建立感染模型；(2)亚急性模型，其中肌肉皮肤感染通过皮下注射孢子悬液诱导。在每个模型中，将评估以下治疗组：血管内皮生长因子、碱性成纤维细胞生长因子、血管内皮细胞生长因子+碱性成纤维细胞生长因子、羊膜移植+血管内皮细胞生长因子、羊膜移植+血管内皮细胞生长因子、羊膜移植+血管内皮细胞生长因子和碱性成纤维细胞生长因子。另外两组小鼠将接受血管生成抑制剂舒尼替尼单独或与AMB联合治疗。三种类型的终点将在治疗组之间进行评估和比较：(1)存活7天(仅限肺模型)；(2)组织真菌负荷，通过定量聚合酶链式反应(QPCR)测量；(3)感染部位的血管生成，使用前面描述的Matrigel分析在活体肌肉皮肤模型中进行评估，并在肺组织切片中使用微血管密度进行评估。在目的2中，我们将确定血管内皮生长因子和碱性成纤维细胞生长因子对AMB组织浓度的影响。将使用高效液相色谱来测量肺组织和Matrigel塞子中的AMB浓度，并在接受AMB单独治疗的小鼠组和接受AMB加血管内皮生长因子和/或碱性成纤维细胞生长因子的小组之间进行比较。公共卫生相关性：侵袭性曲霉病是癌症患者和免疫系统减弱患者患病和死亡的主要原因。尽管有了新的抗真菌药物和改善的支持性护理，但这些感染的死亡率仍然高得令人无法接受。我们推测，侵袭性曲霉病过程中血管的闭塞限制了抗真菌药物对感染组织的渗透，而用促进新血管形成的生长因子治疗可能会改善这种严重的真菌感染的结果。

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.