Intrinsic endothelial hyperresponsiveness to inflammatory mediators drives acute episodes in models of Clarkson disease
Ararat J. Ablooglu, … , Samir M. Parikh, Kirk M. Druey
Ararat J. Ablooglu, … , Samir M. Parikh, Kirk M. Druey
Published March 19, 2024
Citation Information: J Clin Invest. 2024;134(10):e169137. https://doi.org/10.1172/JCI169137.
View: Text | PDF
Research Article Vascular biology

Intrinsic endothelial hyperresponsiveness to inflammatory mediators drives acute episodes in models of Clarkson disease

Abstract

Clarkson disease, or monoclonal gammopathy–associated idiopathic systemic capillary leak syndrome (ISCLS), is a rare, relapsing-remitting disorder featuring the abrupt extravasation of fluids and proteins into peripheral tissues, which in turn leads to hypotensive shock, severe hemoconcentration, and hypoalbuminemia. The specific leakage factor(s) and pathways in ISCLS are unknown, and there is no effective treatment for acute flares. Here, we characterize an autonomous vascular endothelial defect in ISCLS that was recapitulated in patient-derived endothelial cells (ECs) in culture and in a mouse model of disease. ISCLS-derived ECs were functionally hyperresponsive to permeability-inducing factors like VEGF and histamine, in part due to increased endothelial nitric oxide synthase (eNOS) activity. eNOS blockade by administration of N(γ)-nitro-l-arginine methyl ester (l-NAME) ameliorated vascular leakage in an SJL/J mouse model of ISCLS induced by histamine or VEGF challenge. eNOS mislocalization and decreased protein phosphatase 2A (PP2A) expression may contribute to eNOS hyperactivation in ISCLS-derived ECs. Our findings provide mechanistic insights into microvascular barrier dysfunction in ISCLS and highlight a potential therapeutic approach.

Authors

Ararat J. Ablooglu, Wei-Sheng Chen, Zhihui Xie, Abhishek Desai, Subrata Paul, Justin B. Lack, Linda A. Scott, A. Robin Eisch, Arkadiusz Z. Dudek, Samir M. Parikh, Kirk M. Druey

×

Figure 5

Hyperresponsiveness of ISCLS BOECs is eNOS dependent.

Options: View larger image (or click on image) Download as PowerPoint
Hyperresponsiveness of ISCLS BOECs is eNOS dependent. (A) Representative...
(A) Representative immunoblot of relevant receptors or signaling proteins in BOEC cell lysates (n = 5 donors/group). (B) Quantification of relative protein expression. Data indicate the mean ± SEM. n = 6–9 donors/group. NS, by 2-way ANOVA with Šidák’s multiple comparisons. (C) Relative CDH5 or NOS3 expression in BOECs evaluated by quantitative PCR (qPCR) (normalized to Actb and/or GAPDH). Data indicate the mean ± SEM. n = 6–10 donors/group. NS, by Mann-Whitney U test. (D) Relative intracellular Ca2+ concentrations in control (blue) or ISCLS-derived (red) BOECs stimulated with VEGF (100 ng/mL). Data indicate the mean ± SEM. n = 2–3 donors/group analyzed in 4–5 independent experiments. (E) Representative immunoblot showing p-eNOSSer1177 and total eNOS levels in lysates from BOECs stimulated with VEGF and immunoprecipitated with anti-eNOS antibody. (F) Quantification of the p-eNOS/eNOS ratio. Data indicate the mean ± SEM of 3–5 donors/group analyzed in 5 independent experiments. *P = 0.01 and ***P = 0.0008, by 2-way ANOVA with Šidák’s multiple comparisons. (G and H) Representative immunoblot (G) and quantification (H) of eNOS/actin in eNOS siRNA–transfected BOECs. Data indicate the mean ± SEM of 5 independent experiments. ****P < 0.0001, by 1-sample Student’s t test. (I) Representative TER in control or eNOS siRNA–transfected BOECs left untreated (blue and green) or stimulated with VEGF (red and magenta). (J) Maximum decrease in VEGF-induced TER from t = 0 as a percentage of the control siRNA response. Data indicate the mean ± SEM. n = 4–5 donors/group analyzed in 3–5 independent experiments. *P = 0.04, by unpaired, 2-tailed Student’s t test.