Ryanodine Receptors as Therapeutic Targets to Prevent Doxorubicin-Induced Lymphatic Dysfunction

项目总结/摘要 淋巴水肿是乳腺癌和妇科癌症放疗和/或手术后的主要并发症。 手术技术的进步减轻了风险，但水肿的发生率仍然很高， 目前还没有批准的药物来预防或治疗它。多柔比星（DOX）是一种中心化疗药物，用于治疗 乳腺癌和妇科癌症，但它增加了3倍的水肿的风险。的机制 DOX对慢性水肿的作用尚不清楚，但我们发现临床相关的DOX浓度 通过激活兰尼碱受体（RYR， 淋巴肌细胞（LMC）中的细胞内钙通道），导致肌浆紧张性Ca 2+泄漏 网织（SR）和淋巴淤滞。持续高水平的胞浆Ca 2 + [Ca 2 +i]可促进脂质过氧化 和细胞死亡途径，淋巴淤滞引起的腔内压力增加可损害LV 壁和瓣叶，协同引起慢性淋巴损伤。目前尚不清楚DOX是否会激活 RYR通过直接相互作用或通过介导RYR的氧化而间接相互作用。事实上，DOX提升了两者 细胞溶质和线粒体超氧化物（O2·-），这可能有助于RYR氧化（受体开放）， 随后Ca 2+渗漏。也不知道哪种RYR亚型（RYR 1、RYR 2、RYR 3）在D 0X中被DOX激活。 如果已知，它可以作为潜在的治疗靶点，以防止DOX诱导的淋巴功能障碍。 我们提出RYR是LMCs中预防DOX诱导的淋巴功能障碍的新的治疗靶点， 慢性水肿的发展。我们假设DOX产生O2·-， RYR增加LMC中的[Ca 2 +i]，抑制LV收缩并诱导淋巴停滞和淋巴损伤，以及 增加的手术损伤加强了这种作用。因此，我们将使用我们完善的大鼠模型来评估 RYR作为治疗靶标以预防DOX诱导的淋巴功能障碍。三个目标将整合技术 在临床前研究中探索这一假设，并将依赖于分离的LV的蛋白质和功能分析， 使用光学成像评估响应于DOX和RYR阻断的体内淋巴液体积流量，以及 研究RYR阻滞剂的效用，作为淋巴结转移临床前模型中的潜在治疗剂， 不足目的1：探讨DOX诱导的RYR激活是否由O2·-介导。目标2将 定义RYR亚型在分离的LV和体内淋巴流中的DOX诱导的Ca 2+渗漏中的作用。目标3将 研究DOX ± RYR阻断剂对淋巴功能、脂质过氧化和淋巴结转移的联合影响。 在淋巴功能不全的临床前大鼠模型中的形态学。因此，我们计划探索RYRs作为新的 治疗靶点，以预防DOX相关性水肿，并评估RYR阻滞剂是否可用作 抗水肿剂。

PROJECT SUMMARY/ABSTRACT Lymphedema is a major complication after radiation and/or surgery for breast and gynecological cancers. Advancements in surgical techniques mitigate the risk, but the incidence of lymphedema is still high, and there are no approved medications to prevent or treat it. Doxorubicin (DOX) is a central chemotherapy drug for treating breast and gynecological cancers, but it increases the risk of lymphedema by 3-fold. The mechanism by which DOX contributes to chronic lymphedema is unknown, but we found that clinically relevant concentrations of DOX acutely inhibit lymph vessel (LV) contractions and reduce lymph flow by activating ryanodine receptors (RYRs, intracellular calcium channels) in lymph muscle cells (LMCs), resulting in tonic Ca2+ leak from the sarcoplasmic reticulum (SR) and lymphostasis. Sustained high levels of cytosolic Ca2+ [Ca2+i] can promote lipid peroxidation and cell death pathways, and the increase in intraluminal pressure produced by lymphostasis can damage LV walls and valve leaflets, synergistically causing chronic lymphatic injury. It is unclear whether DOX activates RYRs through a direct interaction or indirectly by mediating the oxidation of RYRs. Indeed, DOX elevates both cytosolic and mitochondrial superoxide (O2•-), which could contribute to RYR oxidation (receptor opening) and subsequent Ca2+ leak. It is also unknown which RYR subtype (RYR1, RYR2, RYR3) is activated by DOX in LMCs; if known, it could serve as a potential therapeutic target to prevent DOX-induced lymphatic dysfunction. We propose RYRs are novel therapeutic targets in LMCs to prevent DOX-induced lymphatic dysfunction and the development of chronic lymphedema. We hypothesize that DOX generates O2•- to acutely oxidize and open RYRs to increase [Ca2+i] in LMCs, inhibiting LV contractions and inducing lymphostasis and lymphatic injury, and added surgical insult potentiates this effect. Accordingly, we will use our well-established rat model to evaluate RYRs as therapeutic targets to prevent DOX-induced lymphatic dysfunction. Three aims will integrate techniques in preclinical studies to explore this hypothesis and will rely on protein and functional analysis of isolated LVs, use optical imaging to assess volumetric lymph flow in vivo in response to DOX and RYR blockade, and investigate the utility of RYR blockers, as a potential therapeutics in a preclinical model of lymphatic insufficiency. Aim 1 will determine whether DOX-induced RYR activation is mediated by O2•- in LMCs. Aim 2 will define the role of RYR subtypes in DOX-induced Ca2+ leak in isolated LVs and in lymph flow in vivo. Aim 3 will investigate the combined impact of DOX ± RYR blocker on lymphatic function, lipid peroxidation, and lymphatic morphology in a preclinical rat model of lymphatic insufficiency. Thus, we plan to explore RYRs as novel therapeutic targets to prevent DOX-related lymphedema and evaluate whether RYR blockers can be utilized as anti-lymphedema agents.