Studies in the Pathogenesis of Systemic Capillary Leak Syndrome

全身毛细血管渗漏综合征发病机制的研究

• 批准号: 10014162
• 负责人: Kirk m Druey
• 金额: $ 63.89万
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10014162
• 关键词: Actins; Acute; Address; Adherens Junction; Alleles; Anaphylaxis; Angiogenic Proteins; Angioneurotic Edema; Angiopoietin-2; Animal Model; Antihypertensive Agents; Biological; Biological Assay; Blood; Blood Cells; Blood Vessels; Blood capillaries; CCL2 gene; CXCL10 gene; Candidate Disease Gene; Capillary Leak Syndrome; Cell Line; Clinical; Clinical Protocols; Cytoplasmic Granules; DNA sequencing; Defect; Diagnosis; Disease; Disease remission; Edema; Endothelial Cells; Endothelium; Etiology; Event; Extravasation; Flare; Functional disorder; Genes; Genetic; Genomics; Goals; Growth and Development function; Hematopoietic; Histamine; Human; Hypersensitivity; Hypotension; Hypovolemia; Immune; Immunoglobulin G; Immunoglobulins; Inbred Strains Mice; Infection; Inflammation Mediators; Inflammatory; Interleukin-6; Intravenous Immunoglobulins; Link; Liquid substance; Maps; Mediator of activation protein; Monoclonal gammopathy of uncertain significance; Morbidity - disease rate; Multiple Myeloma; Mus; Mutation; Names; Nature; Neutrophil Collagenase; Odds Ratio; Oliguria; Paraproteins; Pathogenesis; Patients; Penetrance; Permeability; Peroxidases; Plasma; Plasma Cells; Plasmacytic Leukemia; Polymorphism Analysis; Population; Predisposition; Premalignant; Process; Property; Proteins; Proteome; Proteomics; Protocols documentation; RNA; Rare Diseases; Reporting; Research; Role; Sample Size; Sampling; Sepsis; Shock; Signal Pathway; Signs and Symptoms; Single Nucleotide Polymorphism; Skin; Source; Stimulus; Stress Fibers; Susceptibility Gene; Syndrome; Testing; Tissues; United States National Institutes of Health; Variant; Vascular Endothelial Growth Factors; Xenograft Model; acute symptom; anasarca; angiogenesis; aptamer; attenuation; bactericide; base; biomedical referral center; cadherin 5; chemotherapy; cohort; cytokine; density; experience; genetic analysis; genetic association; genome sequencing; genome-wide; immunoregulation; macromolecule; mortality; mouse model; neutrophil; novel therapeutic intervention; response; screening; solute; specific biomarkers; stressor; trait; transcriptome; whole genome

Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A monoclonal gammopathy of unknown significance (MGUS, a premalignant precursor to multiple myeloma (MM), in which a clonal plasma cell population secretes monoclonal immunoglobulin (Ig, also referred to as a paraprotein) detectable in patient sera, is present in a majority of SCLS cases. Several patients with SCLS in whom MGUS evolved into myeloma or plasma cell leukemia experienced fewer capillary leak episodes after chemotherapy for their hematopoietic disorder. These findings suggest that the monoclonal paraprotein from the dysregulated plasma cell population may be the direct or indirect source of the pathophysiologic findings observed. We are characterizing the transcriptome of blood cell RNA and the proteome of SCLS serum/plasma, both pre- and post-attack, to determine whether specific biomarkers of acute symptoms and/or etiological factors can be identified. We have now evaluated more than 70 patients with a confirmed diagnosis of SCLS under this protocol in the last 10 years. We are the primary referral center in the U.S. for SCLS. Circulating permeability factors, vascular endothelial growth factor (VEGF), angiopoietin 2 (Angpt-2), CXCL10, CCL2, and IL-6, were elevated in episodic SCLS sera compared to remission sera. Thus, angiogenic proteins and proinflammatory cytokines that induce endothelial cell (EC) hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. In fiscal year 19, we searched for factors altered in acute SCLS plasma relative to matched convalescent samples using multiplexed aptamer-based proteomic screening. Relative amounts of 612 proteins were changed greater than twofold and 81 proteins were changed at least threefold. Among the most enriched proteins in acute SCLS plasma were neutrophil granule components including bactericidal permeability inducing protein, myeloperoxidase and matrix metalloproteinase 8. Neutrophils isolated from blood of subjects with SCLS or healthy controls responded similarly to routine pro-inflammatory mediators. However, acute SCLS sera activated neutrophils relative to remission sera. Activated neutrophil supernatants increased permeability of endothelial cells from both controls and SCLS subjects equivalently. The transient episodes of hypotensive shock and anasarca in SCLS are thought to arise from reversible microvascular barrier dysfunction. Application of episodic but not convalescent SCLS sera to human microvascular ECs caused vascular endothelial cadherin internalization, disruption of interendothelial junctions, actin stress fiber formation, and increased permeability in complementary functional assays. EC contraction and temporary attenuation of adherens junctions may thus permit leakage of solutes and proteins into the extravascular space during acute episodes. In FY19, we continued to study mechanisms underlying the discovery that the skin microvasculature and endothelial cell lines from SCLS patients are hyper-responsive to routine inflammatory mediators such as VEGF and histamine. Current studies are aimed at identifying abnormalities in the signaling pathways leading to these abnormalities. The role of specific gene defects in SCLS, if any, is unknown; e.g. whether the endothelium is genetically programmed for hyper-responsiveness to routine stimuli. There are no consistent familial aggregations in SCLS. Using Affymetrix Single Nucleotide Polymorphism (SNP) microarrays, we performed the first genome-wide SNP analysis of SCLS in a cohort of 12 disease subjects and 18 controls. From unbiased high-density mapping of single-nucleotide polymorphisms (SNPs), a small genetic interval, 3p25.3, was identified as the highest-ranking candidate susceptibility locus (p 10-6) with an odds ratio of 41. Odds ratios (7-41) and p values (10-4 and 10-6) for the top SCLS-associated variants were outsized for such a small sample size. These results imply high penetrance for a rare disease allele that remains to be identified. We are performing whole genome sequencing of DNA from patient ECs to test the hypothesis that they are prone to exaggerated responses to otherwise mundane inflammatory stressors due to underlying genetic defect(s). In FY19, we continued to develop a mouse model of SCLS. We identified an inbred mouse strain, SJL, that recapitulates cardinal features of SCLS, including susceptibility to histamine- and infection-triggered vascular leak. We named this trait Histamine hypersensitivity (Hhs/Hhs) and mapped it to a region on Chr6. Remarkably, Hhs is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3). These studies reveal that the predisposition to develop vascular hyperpermeability has a strong genetic component conserved between humans and mice and provide a naturally occurring animal model for SCLS. Thus, genetic analysis of the Hhs locus has the potential to reveal and functionally validate orthologous candidate genes that contribute not only to SCLS, but also to normal and dysregulated mechanisms underlying vascular barrier function more generally.

免疫功能失调可能导致SCLS的病理生理学改变。意义不明的单克隆丙种球蛋白病（MGUS，多发性骨髓瘤（MM）的癌前病变，其中克隆浆细胞群分泌可在患者血清中检测到的单克隆免疫球蛋白（IG，也称为副蛋白），存在于大多数SCLS病例中。 几名MGUS演变为骨髓瘤或浆细胞白血病的SCLS患者在化疗后因造血功能障碍发生毛细血管渗漏的次数较少。 这些结果表明，从失调的浆细胞群的单克隆副蛋白可能是直接或间接的来源观察到的病理生理学结果。 我们正在表征血细胞RNA的转录组和SCLS血清/血浆的蛋白质组，包括发作前和发作后，以确定是否可以识别急性症状和/或病因因素的特定生物标志物。在过去的10年里，我们已经评估了70多名确诊为SCLS的患者。我们是SCLS在美国的主要转诊中心。与缓解期血清相比，发作性SCLS血清中的循环渗透因子、血管内皮生长因子（VEGF）、血管生成素2（Angpt-2）、CXCL 10、CCL 2和IL-6升高。因此，诱导内皮细胞（EC）高渗透性的血管生成蛋白和促炎细胞因子可能导致SCLS耀斑周围的短暂EC屏障功能障碍。 在第19财政年度，我们使用基于多重适体的蛋白质组学筛选，搜索了急性SCLS血浆相对于匹配的恢复期样本的改变因素。612种蛋白质的相对量变化超过两倍，81种蛋白质变化至少三倍。急性SCLS血浆中最富集的蛋白质是中性粒细胞颗粒成分，包括杀菌渗透诱导蛋白、髓过氧化物酶和基质金属蛋白酶8。从SCLS受试者或健康对照者血液中分离的中性粒细胞对常规促炎介质的反应相似。然而，急性SCLS血清激活中性粒细胞相对于缓解血清。激活的中性粒细胞上清液增加内皮细胞的渗透性从控制和SCLS受试者等同。 SCLS中短暂发作的水肿性休克和全身水肿被认为是由可逆性微血管屏障功能障碍引起的。在补充功能测定中，将间歇性但非恢复期SCLS血清应用于人微血管EC引起血管内皮钙粘蛋白内化、内皮间连接破坏、肌动蛋白应力纤维形成和通透性增加。因此，EC收缩和粘附连接的暂时衰减可能允许溶质和蛋白质在急性发作期间泄漏到血管外空间中。 在2019财年，我们继续研究SCLS患者的皮肤微血管和内皮细胞系对常规炎症介质（如VEGF和组胺）高度反应的发现背后的机制。目前的研究旨在确定导致这些异常的信号通路中的异常。SCLS中特定基因缺陷的作用（如果有的话）尚不清楚;例如，内皮细胞是否遗传性编程为对常规刺激的高反应性。SCLS中没有一致的家族聚集性。使用Affyssin单核苷酸多态性（SNP）微阵列，我们在12名疾病受试者和18名对照者的队列中进行了SCLS的首次全基因组SNP分析。从单核苷酸多态性（SNPs）的无偏高密度作图，一个小的遗传间隔，3p25.3，被确定为最高级别的候选易感性位点（p10 -6）的优势比为41。 对于如此小的样本量，最高的SCL相关变异的比值比（7-41）和p值（10-4和10-6）过大。这些结果意味着一种罕见疾病等位基因的高等位率仍有待确定。我们正在对来自患者EC的DNA进行全基因组测序，以测试以下假设：由于潜在的遗传缺陷，它们倾向于对原本平凡的炎症应激源产生过度反应。 在2019财年，我们继续开发SCLS小鼠模型。 我们鉴定了一种近交系小鼠品系SJL，它概括了SCLS的主要特征，包括对组胺和感染引发的血管渗漏的易感性。我们将这种特性命名为组胺超敏反应（Hhs/Hhs），并将其映射到Chr 6上的一个区域。值得注意的是，Hhs与人类SCLS最密切相关的基因组位点（3p25.3）同线。这些研究表明，发展血管通透性过高的倾向具有在人类和小鼠之间保守的强遗传成分，并为SCLS提供了自然发生的动物模型。因此，Hhs基因座的遗传分析有可能揭示和功能验证的orthopathy候选基因，不仅有助于SCLS，但也正常和失调的机制，血管屏障功能更普遍。