KATP channels and lymphatic dysfunction in Cantu Syndrome

Cantú综合征(CS)的一个显著特征是超过50%的患者出现某种形式的淋巴水肿。 虽然已知CS的潜在疾病过程涉及KATP的功能获得(GOF)突变 在CS患者中淋巴水肿的发生机制尚不清楚，也尚未得到证实 以前研究过的。因为集合淋巴管的自发收缩是基础？正常的 淋巴运输，我们认为CS中的淋巴水肿，以及潜在的其他形式的淋巴水肿，是由 淋巴管平滑肌兴奋性降低。我们的中心假设是CS相关的GOF突变在 Kir6和/或SuR亚基导致KATP电流过度激活，淋巴管平滑肌超极化 以及抑制驱动自发淋巴收缩的内在电子起搏器。我们有 开创性的方法是使用小鼠的延髓淋巴管来验证我们的假设。直径和指数 在定义的压力/流动条件下，在单个中空容器中测量泵送效率 活着。我们使用尖锐的电极记录淋巴管平滑肌或内皮细胞的膜电位， 并分离和膜片钳任何一种细胞类型，以便选择性地评估KATP电流。在补充输入中 活体研究使用常规或近红外荧光成像，我们评估多瓣膜的功能 使经典的和新型的KATP通道拮抗剂的潜在有益作用可以 接受测试。我们研究小鼠淋巴管的能力使我们能够利用全局和组织- 我们的合作者产生了特定的KATP通道增强型或失调型突变。我们收集了一个 大量的初步数据表明KATP的Kir6.1和SUR2亚型在功能上得到表达 在小鼠淋巴管中，Kir6.1或SUR2GOF小鼠自发淋巴管收缩 严重衰减，但可以通过抑制KATP通道来挽救。这些初步数据是第一次 表现为潜在的收缩缺陷导致原发淋巴水肿。 核心假设将通过三个目标进行检验。1)描述KATP通道的正常调节方式 淋巴抽吸。我们将评估LSM和/或LEC KATP通道在介导抑制 吸入一氧化氮和前列腺素。2)确定CS GOF KATP突变如何损害淋巴管 通过在LEC/LSM层表达突变通道进行泵浦并测量兴奋性和收缩 功能。3)评估CS GOF KATP突变小鼠的淋巴功能障碍能否得到挽救。 已确定的CS淋巴功能障碍的来源(可能是收缩功能缺陷)提供了一个新的机会 探讨药物干预能否有效逆转CS相关的淋巴转运功能障碍。 这些目标的实现将阐明KATP在淋巴管中的作用，揭示 CS的淋巴功能障碍，并测试一种治疗CS和其他患者淋巴水肿的新方法 淋巴管平滑肌兴奋性降低。

A striking feature of Cantú Syndrome(CS) is that over 50% of patients present with some form of lymphedema. Although the underlying disease process in CS is known to involve gain-of-function (GOF) mutations in the KATP channel, the mechanism by which lymphedema develops in CS patients is unknown and has not been studied previously. Because spontaneous contractions of collecting lymphatic vessels underlie ? of normal lymph transport, we propose that lymphedema in CS, and potentially other forms of lymphedema, result from reduced excitability of lymphatic smooth muscle. Our central hypothesis is that CS-related GOF mutations in Kir6 and/or SUR subunits result in hyperactivation of KATP current, hyperpolarization of lymphatic smooth muscle and inhibition of the intrinsic electrical pacemaker that drives spontaneous lymphatic contractions. We have pioneered methods to test our hypothesis using popliteal lymphatic vessels of the mouse. Diameter and indices of pumping efficiency are measured in single, cannulated vessels under defined pressure / flow conditions ex vivo. We record membrane potential in either lymphatic smooth muscle or endothelium using sharp electrodes, and dissociate and patch clamp either cell type in order to selectively assess KATP current. In complementary in vivo studies using conventional or near-infrared fluorescence imaging, we assess the function of multi-valve chains of popliteal vessels so that potentially beneficial effects of classic and novel KATP channel antagonists can be tested. Our ability to study mouse lymphatic vessels allows us to take advantage of the global and tissue- specific KATP channel gain- or loss-of-function mutants generated by our collaborators. We have collected a substantial amount of preliminary data to show that Kir6.1 and SUR2 isoforms of KATP are functionally expressed in mouse lymphatic vessels and that spontaneous lymphatic contractions in Kir6.1 or SUR2 GOF mice are severely attenuated but can be rescued by inhibition of KATP channels. These preliminary data are the first demonstration of an underlying contractile defect leading to primary lymphedema. The central hypothesis will be tested with 3 aims. 1) Delineate how KATP channels normally regulate lymphatic pumping. We will assess the roles of LSM and/or LEC KATP channels in mediating inhibition of pumping by nitric oxide and prostanoids. 2) Determine how CS GOF KATP mutations impair lymphatic pumping by expressing the mutant channels in the LEC / LSM layers and measuring excitability and contractile function. 3) Assess whether lymphatic dysfunction can be rescued in mice with CS GOF KATP mutations. The identified source of lymphatic dysfunction in CS (a possible contractility deficit) provides a novel opportunity to test if pharmacologic intervention can be effective in reversing CS-related lymph transport dysfunction. Accomplishment of these aims will elucidate the role of KATP in lymphatic vessels, uncover the mechanism of lymphatic dysfunction in CS, and test a novel therapeutic method to treat lymphedema in CS and other patients with reduced lymphatic smooth muscle excitability.