Studies in the Pathogenesis of Systemic Capillary Leak Syndrome

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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 65 patients with a confirmed diagnosis of SCLS under this protocol in the last 9 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 EC hyper-permeability may contribute to transient EC barrier dysfunction around SCLS flares. In FY18, we extended these findings by performing a comprehensive proteomic screen (1300 proteins) of acute and convalescent sera from SCLS patients using Somalogics technology. This study revealed transient deflections of adrenomedullin, a hormone derived primarily from monocytes and endothelial cells (ECs) during acute SCLS attacks. Adrenomedullin promotes vascular smooth muscle relaxation and endothelial barrier integrity. Monocytes from SCLS patients expressed more adrenomedullin than those from healthy controls. An additional finding of this study was the presence of neutrophil granule proteins, suggesting that neutrophils undergo activation during acute flares of 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 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 FY18, we extended these initial findings to demonstrate 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 permeability that could account for 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 FY18, we utilized an inbred mouse strain to develop a model of SCLS. SJL/J mice have circulating paraproteins similar to MGUS in human SCLS, and they exhibit a propensity for vascular leak-induced mortality after systemic administration of histamine. This trait mapped to a recessive locus on mouse chromosome 6 syntenic with human 3p25.3, which harbors the primary genetic association with SCLS in humans. Approximately 50% of patients with SCLS have antecedent infections (typically viral) that trigger acute SCLS attacks. We found that SJL mice but not other inbred strains exhibit vascular leak, primarily in skin and skeletal muscle, following influenza infection. Current studies are aimed at narrowing the genetic interval responsible for the "SCLS-like" phenotype in order to identify specific gene candidates that can be interrogated in functional complementation studies.