Altered Lymphatic Function and Development in Congenital Heart Disease

项目摘要/摘要 淋巴结构和功能的异常日益被认为是发病率的一个重要来源 在各种疾病状态下。例如，与先天性心脏病有关的淋巴异常 (CHD)导致肺血流量(PBF)增加，包括先天性或获得性乳糜胸，以及 免疫异常。重要的是，人们越来越认识到淋巴异常不仅是 与多种肺部疾病相关，但参与其发病机制。最近认识到 异常呼吸功能障碍通常在心脏矫形术后数年持续存在，是一个独立的危险因素。 因为成年冠心病患者的死亡率再次带来了更好地了解潜在淋巴管的紧迫性 冠心病的病理生物学，到目前为止，这在很大程度上仍然是未知的。我们之前已经使用了一种临床相关的 PBF(分流)增加的CHD大动物模型结合血管离体反应性 胸导管和原代淋巴管内皮细胞(LEC)培养，以证明慢性PBF增加 随之而来的淋巴流量的增加与：1)肺淋巴流量异常和 结构；2)线粒体活性氧种(MtROS)驱动的缺氧诱导因子-1增加 (HIF-1)活性和代谢重编程以支持细胞和增殖；以及3)依赖KLF2 一氧化氮(NO)信号的减少。在此续订应用程序中，我们将测试我们的新假设 在PBF增加的情况下，机械感觉通道Piezo1在感知脑缺血后的改变中起着关键作用 淋巴流动，触发下游内皮素-1(ET-1)和mtROS驱动的HIF-1a活性增加。这 导致NO生物利用度降低和随后的淋巴功能障碍，这有助于持续 即使在心脏缺陷被修复后，也会出现呼吸力学异常。为了支持这一假设， 我们提供的初步证据表明：1)淋巴管内皮细胞Piezo1、ET-1、RhoA和 分流性LEC中的mtROS；2)对照LEC中依赖Piezo1、ET-1和mtROS的HIF-1的增加；3)分流性LECs 羔羊有异常的呼吸力学和淋巴管内皮细胞功能障碍，在关闭后持续存在 4)使用线粒体靶向抗氧化剂治疗 (Mitoquone，MitoQ)体外逆转HIF-1介导的分流性晶状体上皮细胞表型并使肺功能正常化 体内淋巴的结构和功能。这一总体假设将在三个相互关联的领域进行检验，但 独立的机械学目标，利用综合的生理学、细胞和分子实验。这些 转化性和针对性研究包括：1)全动物血流动力学生理学研究，高级 CT/MR成像和复杂的肺功能测试，2)体外胸管反应性研究，3) 晶状体上皮细胞的转录和代谢研究。对所调查的机制有更深入的了解可能会导致 改进对冠心病和其他疾病患者淋巴异常的治疗和预防策略 疾病状态，包括肺切除和其他导致PBF增加的血管异常。

PROJECT SUMMARY / ABSTRACT Aberrations in lymphatic structure and function are increasingly recognized as a significant source of morbidity in a variety of disease states. For example, lymphatic abnormalities associated with congenital heart disease (CHD) that result in increased pulmonary blood flow (PBF) include congenital or acquired chylothoraces, and immunologic aberrations. Importantly, it is increasingly appreciated that lymphatic abnormalities are not only associated with a variety of pulmonary diseases but participate in their pathogenesis. Recent recognition that abnormal respiratory dysfunction often persists years after cardiac correction and is an independent risk factor for mortality in adult CHD patients has brought renewed urgency to better understand the underlying lymphatic pathobiology in CHD, which thus far remains largely unknown. We have previously used a clinically relevant large animal model of CHD with increased PBF (shunt) in combination with isolated vessel reactivity of the thoracic duct and primary lymphatic endothelial cell (LEC) culture, to demonstrate that chronically increased PBF and the consequential increase in lymphatic flow, is associated with: 1) abnormal pulmonary lymphatic flow and architecture; 2) increased mitochondrial reactive oxygen species (mtROS)-driven hypoxia inducible factor-1 (HIF-1) activity and metabolic reprograming to support cellular and proliferation; and 3) a KLF2-dependent decrease in nitric oxide (NO) signaling. In this renewal application we will test our novel hypothesis that in the setting of increased PBF, the mechanosensory channel Piezo1 plays a pivotal role sensing alterations in lymphatic flow, triggering downstream increases in endothelin-1 (ET-1) and mtROS driven HIF-1a activity. This results in decreased NO bioavailability and subsequent lymphatic dysfunction, that contributes to persistently abnormal respiratory mechanics even after the cardiac defect has been repaired. In support of this hypothesis, we present preliminary evidence demonstrating: 1) increased lymphatic endothelial Piezo1, ET-1, RhoA, and mtROS in shunt LECs; 2) Piezo1-, ET-1-, and mtROS-dependent increases in HIF-1 in control LECs; 3) shunt lambs have abnormal respiratory mechanics and lymphatic endothelial dysfunction that persists following closure of the shunt, despite normalized hemodynamics, and 4) that treatment with a mitochondrially-targeted antioxidant (mitoquinone, MitoQ) reverses the HIF-1 mediated shunt LEC phenotype in vitro and normalizes pulmonary lymphatic architecture and function in vivo. This overall hypothesis will be tested in three inter-related, but independent mechanistic aims, that utilize integrated physiologic, cellular, and molecular experiments. These translational and targetable studies include: 1) whole animal hemodynamic physiologic studies, advanced CT/MR imaging, and sophisticated pulmonary function testing, 2) ex vivo thoracic duct reactivity studies, and 3) transcriptional and metabolic studies in LECs. A deeper understanding of the mechanisms investigated may lead to improved treatment and prevention strategies for lymphatic abnormalities in the setting of CHD and other disease states, including pneumonectomy and other vascular abnormalities that result in increased PBF.