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。多发性骨髓瘤(MM)的癌前病变(MGUS)是一种未知意义的单克隆性丙种球蛋白(MGUS)，在大多数SCLS病例中，克隆性浆细胞群分泌在患者血清中可检测到的单克隆性免疫球蛋白(Ig，也称为副蛋白)。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。 我们正在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鉴定，以确定是否可以识别急性症状和/或病因的特定生物标记物。在过去的8年里，我们已经评估了63名在该方案下确诊为SCLS的患者。我们是SCLS在美国的主要转介中心。 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(ECs)，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。 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。因此，诱导内皮细胞高通透性的血管生成蛋白和促炎细胞因子可能是导致小细胞淋巴瘤斑块周围一过性内皮屏障功能障碍的原因。 为了测试Angpt2途径和/或其他通透性诱导剂对SCLS的可能贡献，我们从静脉穿刺法获得的循环前体细胞中扩增出血液生长内皮细胞(BOECs)。这些细胞具有成熟内皮细胞的结构和分子特征。初步结果表明，与健康对照组相比，SCLS BOECs中的基因表达模式异常。目前的研究旨在检测来自SCLS患者和对照组的内皮细胞对各种通透性介质的反应，以验证SCLS内皮细胞容易对其他普通炎症应激源做出夸大反应的假设。 特定基因缺陷在SCLS中的作用也是未知的；例如，内皮细胞是否具有对常规刺激的高反应性。在SCL中没有一致的家族聚集性。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。 从这些阵列中发现的最常见的是多聚ADP核糖基转移酶15(PARP15)中的SNP，它只在患者组中存在，而在对照组中没有。NIH队列的完整外显子测序和Sanger测序显示，该SNP在SCLS中的频率是对照组的两倍，包括来自ExAc或1000基因组数据库的受试者。生化研究表明，该突变导致PARP15酶活性部分丧失。CRISPR对微血管内皮细胞中PARP15基因的敲除揭示了PARP15调节通透性的途径。因此，PARP15突变可能在SCLS的发病机制中起一定作用。目前，我们正在研究这种表型背后的分子机制。 鉴定了一种近交系小鼠(SJL/J)，其表型与SCLS相似，包括对多种通透性因子(包括组胺、5-羟色胺和缓激肽)的自发血管超敏反应，这些通透性因子是人类内皮高通透性的重要介质，以及以血清副蛋白存在为特征的B细胞肿瘤。在SJL小鼠中，一个控制全身血管对透明质酸超敏反应(SHS)的隐性基因被定位在小鼠6号染色体上与人类3p25.3同线的区间，该基因与人类的SCLS具有主要的遗传关联。这个区间包含几个候选基因，将在SCLS队列和各种小鼠品系中进行分析。

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 63 patients with a confirmed diagnosis of SCLS under this protocol in the last 8 years. We are the primary referral center in the U.S. for SCLS. 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 endothelial cells (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. 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 EC hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. To test the possible contribution of the Angpt2 pathway and/or other permeability inducers to SCLS, we have expanded blood-outgrowth ECs (BOECs) from circulating precursors obtained by venipuncture. These cells possess structural and molecular characteristics of mature ECs. Preliminary results suggest abnormal gene expression patterns in SCLS BOECs compared to those from healthy controls. Current studies are aimed at examined responses of ECs derived from subjects with SCLS and controls to various mediators of permeability to test the hypothesis that the SCLS endothelium is prone to exaggerated responses to otherwise mundane inflammatory stressors. The role of specific gene defects in SCLS, if any, is also 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. A top hit from these arrays was a SNP in poly-ADP ribosyltransferase 15 (PARP15), which was present exclusively in the patient group but none of the controls. Whole exome sequencing and Sanger sequencing of the NIH cohort revealed that the frequency of this SNP in SCLS was double that of controls, including subjects from databases such as ExAc or 1000 Genomes. Biochemical studies showed that this mutation resulted in a partial loss of PARP15 enzyme activity. Knockout of PARP15 in microvascular endothelial cells by CRISPR revealed that PARP15 regulates permeability pathways. As such, PARP15 mutations may have a role in the pathogenesis of SCLS. Currently we are examining the molecular mechanisms underlying this phenotype. An inbred mouse strain was identified (SJL/J), whose phenotype bears similarity to SCLS, including spontaneous vascular hypersensitivity to a variety of permeability factors including histamine (HA), serotonin, and bradykinin, important mediators of endothelial hyperpermeability in humans, and B cell neoplasms characterized by the presence of serum paraproteins. A recessive locus in SJL mice controlling systemic vascular hypersensitivity to HA (Shs) was mapped to an interval on mouse chromosome 6 syntenic with human 3p25.3, which harbors the primary genetic association with SCLS in humans. This interval contains several gene candidates, which will be analyzed in the SCLS cohort and various mouse strains.