Studies in the Pathogenesis of Systemic Capillary Leak Syndrome

免疫失调可能是导致SCLS病理生理改变的原因之一。MGUS是多发性骨髓瘤(MM)的癌前病变，是一种以分泌单克隆性免疫球蛋白(Ig，也称为副蛋白)的克隆性浆细胞群为特征的疾病。MGUS在大多数SCLS病例中均可检出。几名小细胞淋巴瘤患者中，MGUS进展为骨髓瘤或浆细胞白血病的患者在化疗后因其造血功能障碍而出现较少的毛细血管渗漏事件。这些发现表明，来自失调浆细胞群体的单克隆性副蛋白可能是观察到的病理生理结果的直接或间接来源。目前的研究主要集中在利用高通量蛋白质阵列鉴定SCLS单抗Ig的靶抗原。 我们还对发病前和发病后的血细胞RNA转录组和SCLS血清/血浆的蛋白质组进行了鉴定，以确定是否可以识别急性SCLS症状和/或病因的特定生物标志物。在过去的11年里，我们已经根据这一方案评估了近75名确诊为SCLS的患者。我们是SCLS在美国的主要转介中心。与缓解期血清相比，SCLS发作期血清循环通透性因子血管内皮生长因子(VEGF)、血管生成素-2(Angpt-2)、CXCL10、CCL2和IL-6升高。这些发现提示，诱导内皮细胞(EC)高通透性的血管生成蛋白和促炎细胞因子可能是SCLS耀斑周围一过性EC屏障功能障碍的原因之一。 SCLS中的一过性低血压休克和无症状性发作被认为是可逆性微血管屏障功能障碍引起的。将间歇性但非恢复期的SCLS血清应用于人微血管内皮细胞，可导致血管内皮细胞钙粘附素内化，内皮细胞间连接中断，肌动蛋白应激纤维形成，并在补充功能分析中增加通透性。因此，在急性发作期间，内皮细胞的收缩和附着连接的暂时减弱可能会使溶质和蛋白质泄漏到血管外间隙。在20财年，我们继续研究SCLS患者皮肤微血管和内皮细胞系对常规炎症介质(如血管内皮生长因子和组胺)高度反应的机制。目前的研究旨在确定这些异常背后的信号通路中的分子缺陷。 特定基因缺陷在SCLS中的作用尚不清楚；例如，内皮细胞是否具有对常规刺激的高反应性。在SCLS中没有一致的家族聚集性，对无关的SCLS成人和几个亲子三人组的全外显子组测序未能揭示统一的外来病因学。使用Affymetrix单核苷酸多态(SNP)微阵列，我们首次对12名疾病受试者和18名对照的SCLS进行了全基因组SNP分析。从单核苷酸多态的无偏高密度图谱(SNPs)中，一个小的遗传区间3p25.3被确定为排名最高的候选易感基因座(p10-6)，优势比为41。对于如此小的样本量，顶级SCLS相关变种的优势比(7-41)和p值(10-4和10-6)都过大了。这些结果表明，一种罕见疾病的等位基因有很高的外显率，尚待鉴定。目前，我们正在对患者的脱氧核糖核酸进行全基因组测序，以检验这一假设，即由于潜在的遗传缺陷，他们容易对其他普通的炎症应激因素产生夸大反应(S)。 在2020财年，我们描述了SCLS的原始啮齿动物模型。我们鉴定了一种近交系小鼠，SJL，它概括了SCLS的基本特征，包括对组胺和感染引发的血管泄漏的易感性。我们将这一性状命名为组胺高敏感性，并将其定位在6号染色体上。组胺高敏基因与人类SCLS(3p25.3)密切相关，揭示了发生血管高通透性的易感性具有强烈的遗传成分，在人类和小鼠之间保守，为SCLS提供了一个自然发生的动物模型。对HITH的遗传分析可能揭示出同源候选基因，这些基因不仅对SCLS有贡献，而且对更广泛的血管屏障功能的正常和失调机制也有贡献。 最后，我们与PharmAbine，Inc.合作，研究一种针对Angpt2受体Tie2的单抗PMC-403。PMC-403是一种新型的激动型抗体，能与人Tie2受体结合，促进小鼠肿瘤模型中泄漏血管的稳定。这一关键发现最近在2020年美国癌症研究协会(AACR)会议上公布，预计PMC-403将于2022年进入I期临床试验。我们将研究PMC-403在临床前小鼠SCLS模型中的作用及其对体外培养的SCLS内皮细胞炎性介质的影响。

Immune dysregulation may contribute to the pathophysiologic findings seen in SCLS. A MGUS, a premalignant precursor to multiple myeloma (MM), is a condition characterized by a clonal plasma cell population that secretes monoclonal immunoglobulins (Ig, also referred to as a paraprotein). MGUS is detectable 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. Current studies are focused on identifying the target antigen of SCLS monoclonal Ig, if any, using high-throughput protein arrays. We are also 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 SCLS symptoms and/or etiological factors can be identified. We have now evaluated nearly 75 patients with a confirmed diagnosis of SCLS under this protocol in the last 11 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. These findings suggest that angiogenic proteins and proinflammatory cytokines that induce endothelial cell (EC) hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. 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 fiscal year 20, 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 molecular defects in the signaling pathways that underly 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, and whole exome sequencing of unrelated adults with SCLS and several parent-child trios failed to reveal a uniform exotic etiology. 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. Currently, we are performing whole genome sequencing of DNA from patients to test the hypothesis that they are prone to exaggerated responses to otherwise mundane inflammatory stressors due to underlying genetic defect(s). In FY20, we described an original rodent model of SCLS. We identified an inbred mouse strain, SJL, which recapitulates cardinal features of SCLS, including susceptibility to histamine- and infection-triggered vascular leak. We named this trait Histamine hypersensitivity (Histh/Histh) and mapped it to Chromosome 6. Histh is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3), revealing that the predisposition to develop vascular hyperpermeability has a strong genetic component conserved between humans and mice and providing a naturally occurring animal model for SCLS. Genetic analysis of Histh may reveal orthologous candidate genes that contribute not only to SCLS, but also to normal and dysregulated mechanisms underlying vascular barrier function more generally. Finally, we entered into a collaboration with PharmAbcine, Inc. to study a monoclonal antibody, PMC-403, which targets the Angpt2 receptor, Tie2. PMC-403 is a novel agonistic antibody that binds to human-Tie2 receptor and promotes stabilization of leaky blood vessels in tumor models in mice. The key findings were presented recently at the American Association of Cancer Research (AACR) meeting 2020, and PMC-403 is expected to enter a phase I clinical trial in 2022. We will investigate PMC-403 in the preclinical mouse model of SCLS and its effects on SCLS ECs to inflammatory mediators in vitro.