Non-Invasive Methods for Imaging Myocardial Angiogenesis

该项目的目标是开发非侵入性成像策略来识别血管生成的低氧刺激，并使用靶向成像来无创性地跟踪缺血模型中血管生成过程随时间的变化与局部心肌灌注和低氧的关系。已知心肌缺氧可刺激心脏血管生成，从而为血管生成的启动提供一个潜在的标记物。我们假设有效的血管生成应该导致静息心肌灌注的改善和局部心肌缺氧的减少。我们假设~(99m)Tc-硝基咪唑单光子发射计算机断层扫描(SPECT)可以无创性地识别心肌缺氧。血管生成反应也受细胞外基质的组成和细胞间黏附的调节。我们还假设，通过针对血管生成标记物的放射性标记配体的SPECT成像，可以直接非侵入性地跟踪血管生成过程，如αvbeta3整合素。这些假设将在四个具体目标中得到解决。目的1研究99mTc标记的硝基咪唑在体外和体内制剂中的滞留与缺氧程度的关系。缺氧的程度也与缺氧和血管生成的急性标志物上调有关。目的2研究标记的喹诺酮类αvbeta3拮抗剂对血管生成的选择性。利用体外血管内皮细胞培养，将放射性标记拮抗剂的荧光标记类似物与高度特异的αvbeta3抗体(LM609)进行比较。目的3利用已建立的啮齿动物血管生成实验模型，研究局部缺氧和血流灌注的时间变化与放射性标记的aphavbeta3拮抗剂局部心肌摄取变化的关系。局部缺氧、血流和整合素表达的改变将与血管生成的免疫组织化学和免疫荧光标记物有关。为了将这些发现从大鼠模型扩展到临床可应用的非侵入性成像策略，Aim 4将评估SPECT成像直接跟踪导管诱导再灌注梗塞慢性犬模型血管生成过程的能力。我们将比较使用111In标记的αvbeta3拮抗剂和99mTc标记的硝基咪唑的SPECT成像策略，以及放射性标记微球测量局部心肌流量、201T1灌注和血管生成的免疫组织学分析。为了实现这一最终目标，将进行更多的心脏模体研究，以开发和验证双同位素灌注和99mTc或111In标记的血管生成“热点”SPECT图像的定量分析方法。所提出的核成像方法旨在利用血管生成的特定生物学来提供至关重要的非侵入性方法来评估患者的血管生成。为评估患者血管生成的方法开发更灵敏和特异的成像策略。发展更灵敏和特异的评价心肌血管生成的成像策略，并无创地跟踪这一过程，对于评价新的心脏血管生成疗法至关重要。

The goal of this project is to develop non-invasive imaging strategies for identifying the hypoxic stimulus for angiogenesis, and to use targeted imaging to non-invasively track the angiogenic process over time in ischemic models in relationship to changes in regional myocardial perfusion, and hypoxia. Myocardial hypoxia is known to stimulate angiogenesis in the heart, thereby providing a potential marker for the initiation of angiogenesis. We hypothesize that effective angiogenesis should result in an improvement in resting myocardial perfusion and a reduction in regional myocardial hypoxia. We hypothesize that myocardial hypoxia can be identified non-invasively with 99mTc-nitroimidazole single photon emission computed tomography (SPECT) imaging. The angiogenic response is also controlled by modulation of the composition of the extracellular matrix and intercellular adhesions. We also hypothesize that the angiogenic process can be directly tracked non-invasively by SPECT imaging of radiolabeled ligands targeted to markers of angiogenesis, like the alphavbeta3 integrin. These hypotheses will be addressed in 4 specific aims. Aim 1 will investigate the relationship between level of hypoxia and retention of a 99mTc-labeled nitroimidazole using in vitro, and in vivo preparations. The degree of hypoxia will also be related to the up-regulation of acute markers of hypoxia and angiogenesis. Aim 2 will investigate the selectivity of a labeled quinolone alphavbeta3 antagonist for identification of angiogenesis. Localization of a fluorescent-labeled analog of the radiolabeled antagonist will be compared with a highly specific alphavbeta3 antibody (LM609), using in vitro endothelial cell cultures. Aim 3 will investigate the temporal changes in regional hypoxia and perfusion in relationship to changes in regional myocardial uptake of the radiolabeled aphavbeta3 antagonist, using established experimental rodent models of angiogenesis. Alterations in regional hypoxia, perfusion and integrin expression will be related to immuno-histochemical and immuno-fluorescent markers of angiogenesis. To extend the findings from the rat models towards clinically applicable non- invasive imaging stategies, Aim 4 will evaluate the ability of SPECT imaging to directly track the angiogenic process in chronic canine models of catheter induced reperfused infarction. We will compare SPECT imaging strategies employing the 111In-labeled alphavbeta3 antagonist and 99mTc-labeled nitroimidazole, with radiolabeled microsphere measures of regional myocardial flow, 201T1 perfusion, and immuno-histological analysis of angiogenesis. To accomplish this final aim, additional cardiac phantoms studies will be performed to develop and validate approaches for quantitative analysis of dual isotope perfusion and 99mTc-or 111 In-labeled "hot spot" SPECT images of angiogenesis. The proposed nuclear imaging approaches are designed to exploit the specific biology of angiogenesis to provide critically important non-invasive methods of evaluating angiogenesis in patients. The development of more sensitive and specific imaging strategies for evaluation of methods of evaluating angiogenesis in patients. The development of more sensitive and specific imaging strategies for evaluation of myocardial angiogenesis and tracking this process non-invasively are critical in evaluation of new angiogenic therapy for the heart.