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 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.