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

项目摘要/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。 手术技术的进步降低了风险，但淋巴水肿的发生率仍然很高，而且 没有经过批准的药物来预防或治疗它。阿霉素(DOX)是一种中枢化疗药物 乳腺癌和妇科癌症，但它会使淋巴水肿的风险增加3倍。这一机制通过它 DOX对慢性淋巴水肿的作用尚不清楚，但我们发现临床上相关浓度的DOX 通过激活兰尼定受体(RYRs)，显著抑制淋巴管(LV)收缩，减少淋巴流量 细胞内钙通道)，导致肌浆紧张性钙泄漏 网状(SR)和淋巴滞留。持续高水平的细胞内钙[钙离子]i可促进脂质过氧化 和细胞死亡途径，淋巴滞留引起的腔内压力增加可以损害左心室。 壁和瓣叶，协同作用造成慢性淋巴损伤。目前尚不清楚DOX是否激活 RYRs通过直接相互作用或间接通过调节RYRs的氧化来实现。事实上，DOX提高了这两个指标 胞浆和线粒体超氧化物(O2·-)，这可能有助于RyR氧化(受体开放)和 随后的钙离子泄漏。也不知道DOX在哪种RyR亚型(RYR1、RYR2、RYR3)被激活 LMCs；如果已知，它可能成为预防DOX诱导的淋巴功能障碍的潜在治疗靶点。 我们认为RYRs是预防DOX诱导的淋巴功能障碍的新的治疗靶点，并且 慢性淋巴水肿的发展。我们假设DOX产生O2·-来剧烈地氧化和打开 RYRs增加LMCs内[Ca2+i]，抑制LV收缩，导致淋巴滞留和淋巴损伤，以及 再加上外科手术的侮辱，就会加剧这种影响。因此，我们将使用我们成熟的大鼠模型来评估 RYRs作为预防DOX所致淋巴功能障碍的治疗靶点。三个目标将整合技术 在探索这一假说的临床前研究中，将依赖于对分离的LV的蛋白质和功能分析， 使用光学成像来评估体内对DOX和RyR阻断的反应的体积淋巴流量，以及 研究RyR阻滞剂在淋巴系统临床前模型中作为潜在治疗药物的有效性 不够用。目的1确定DOX诱导的肺小管上皮细胞RyR激活是否由O2·-介导。目标2将 明确RyR亚型在DOX诱导的离体LV钙渗漏和体内淋巴流动中的作用。目标3将 研究DOX±RyR阻滞剂对淋巴功能、脂质过氧化和淋巴的联合影响 临床前淋巴管功能不全大鼠模型的形态。因此，我们计划将RYR作为小说来探索 预防DOX相关性淋巴水肿的治疗目标和评估RyR阻滞剂是否可用于 抗淋巴水肿剂。

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.