Mechanism and Prevention of Doxorubicin-Induced Lymphedema

项目总结/摘要 淋巴水肿是乳腺癌放疗和/或手术后的主要并发症。可惜 严重的并发症包括疼痛、淋巴管炎、蜂窝织炎、溃疡和 恶性淋巴管瘤的发展。多柔比星（DOX）是一种中枢化疗剂， 治疗乳腺癌，但它会增加水肿的风险4至5倍。DOX的作用机制 导致水肿的发展尚不清楚，但认为与其细胞毒性作用有关。 然而，我们认为临床浓度的DOX直接抑制淋巴的节律性收缩， 淋巴管（LV）将淋巴液从组织推回心脏，以防止水肿。这些收缩 受到LV中钙浓度和氧化还原状态的严格控制。高分辨率体内成像还 揭示了全身给予DOX显著减少淋巴流量。抑制左心室收缩 Dantrolene（DANT）是一种临床上可用的兰尼定阻滞剂， 受体（RYR）和线粒体特异性活性氧（mitoROS）清除剂MitoTEMPO。 这些数据，沿着DOX激活RYR并诱导纹状体细胞线粒体功能障碍的知识， 肌肉和最近在LV中发现的功能性RYR，使我们假设：DOX急性开放 RYR增加淋巴肌细胞（LMC）中的细胞溶质钙和线粒体ROS，导致细胞内钙离子的损失。 LV收缩并诱导淋巴淤滞和淋巴损伤。因此，我们将探讨是否 DANT是FDA批准的RYR阻滞剂，可以预防DOX诱导的淋巴功能障碍。三个目标将 使用技术的整合来探索这一假设，并将依赖于蛋白质和功能分析， 分离的淋巴肌细胞和整个LV，使用光学成像来评估体内的体积淋巴流量， 对DOX和RYR阻断的反应，并研究DANT作为大鼠乳腺癌的潜在治疗剂的效用。 肿瘤模型目的1将定量RYR亚型的基因和蛋白表达谱， DOX是否激活RYR以增加细胞溶质钙和线粒体ROS的产生作为一种机制， 抑制左心室收缩目标2将评估DANT是否代表预防DOX的潜在治疗选择- 诱导的淋巴流动抑制和防止DOX诱导的淋巴损伤。目标3将研究 DANT对DOX在乳腺癌大鼠模型中的抗癌活性的影响。因此，我们计划探索RYR， DOX相关性水肿的新治疗靶点，并评估RYR阻滞剂是否可以重新利用 作为抗水肿药物。

PROJECT SUMMARY/ABSTRACT Lymphedema is a major complication after radiation and/or surgery for breast cancer. Unfortunately, there are no medications to effectively treat it. Serious complications include pain, lymphangitis, cellulitis, ulcers, and the development of malignant lymphangiosarcomas. Doxorubicin (DOX) is a central chemotherapeutic agent for treating breast cancer, but it increases the risk of lymphedema by 4- to 5-fold. The mechanism by which DOX contributes to the development of lymphedema is unknown, but it is thought to relate to its cytotoxic action. However, we propose that clinical concentrations of DOX directly inhibit the rhythmic contractions of lymph vessels (LVs) that propel lymph fluid from tissues back to the heart to prevent lymphedema. These contractions are tightly controlled by the calcium concentration and redox state in LVs. High-resolution in vivo imaging also reveals that systemically administered DOX profoundly reduces lymph flow. The suppression of LV contractile function by DOX is largely prevented by both dantrolene (DANT), a clinically available blocker of ryanodine receptors (RYRs), and MitoTEMPO, a mitochondrial-specific reactive oxygen species (mitoROS) scavenger. These data, along with knowledge that DOX activates RYRs and induces mitochondrial dysfunction in striated muscle and recent discoveries of functional RYRs in LVs, have led us to hypothesize that: DOX acutely opens RYRs to increase cytosolic calcium and mitoROS in lymph muscle cells (LMCs), resulting in the loss of LV contractions and inducing lymphostasis and lymphatic injury. Accordingly, we will explore whether DANT, an FDA-approved RYR blocker, can prevent DOX-induced lymphatic dysfunction. Three aims will use an integration of techniques to explore this hypothesis and will rely on protein and functional analysis of isolated lymph muscle cells and whole LVs, use optical imaging to assess volumetric lymph flow in vivo in response to DOX and RYR blockade, and investigate the utility of DANT as a potential therapeutic in a rat breast tumor model. Aim 1 will quantify the gene and protein expression profiles of RYR subtypes and determine whether DOX activates RYRs to increase cytosolic calcium and mitoROS generation as a mechanism of inhibiting LV contractions. Aim 2 will evaluate if DANT represents a potential therapeutic option to prevent DOX- induced suppression of lymph flow and prevent DOX-induced lymphatic injury. Aim 3 will investigate the effects of DANT on the anticancer activity of DOX in a rat model of breast cancer. Thus, we plan to explore RYRs as new therapeutic targets for DOX-related lymphedema and evaluate whether RYR blockers can be repurposed as anti-lymphedema medications.