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水肿。 尽管已知CS的潜在疾病过程涉及KATP中的功能获得性（GOF）突变， 通道，在CS患者中发生水肿的机制尚不清楚， 以前研究过。因为集合淋巴管的自发收缩是？正常 淋巴转运，我们认为CS中的水肿和潜在的其他形式的水肿是由 淋巴管平滑肌的兴奋性降低。我们的中心假设是，CS相关的GOF突变， Kir 6和/或SUR亚基导致KATP电流过度激活、淋巴平滑肌超极化 以及抑制驱动自发淋巴收缩的内在电起搏点。我们有 开创性的方法来测试我们的假设，使用小鼠的腘淋巴管。直径和指数 泵送效率的测量是在单一的，空心血管在规定的压力/流量条件下， vivo.我们用锋利的电极记录淋巴管平滑肌或内皮细胞的膜电位， 并解离和膜片钳任一细胞类型，以选择性地评估KATP电流。在互补中， 使用常规或近红外荧光成像的体内研究，我们评估了多瓣膜的功能， 因此经典和新型KATP通道拮抗剂的潜在有益作用可以 得到考验我们研究小鼠淋巴管的能力使我们能够利用全球和组织- 我们的合作者产生的特定KATP通道功能获得或丧失突变体。我们收集了 大量初步数据显示KATP Kir6.1和SUR 2同种型在功能上表达 Kir6.1或SUR 2 GOF小鼠中自发性淋巴收缩 严重减弱，但可通过抑制KATP通道来挽救。这些初步数据是 证明潜在的收缩缺陷导致原发性水肿。 中心假设将通过3个目标进行检验。1)描述KATP通道如何正常调节 淋巴泵我们将评估LSM和/或LEC KATP通道在介导抑制细胞凋亡中的作用。 由一氧化氮和前列腺素泵送。2)确定CS GOF KATP突变如何损害淋巴 通过在LEC / LSM层中表达突变通道并测量兴奋性和收缩性， 功能3)评估是否可以在具有CS GOF KATP突变的小鼠中挽救淋巴功能障碍。 CS中淋巴功能障碍的确定来源（可能的收缩性缺陷）提供了一个新的机会 以测试药物干预是否能有效逆转CS相关的淋巴转运功能障碍。 这些目标的实现将有助于阐明KATP在淋巴管中的作用，揭示KATP在淋巴管中的作用机制， 淋巴功能障碍，并测试一种新的治疗方法来治疗CS和其他患者的水肿 淋巴平滑肌兴奋性降低

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.