Citrullinated histone 3 causes endothelial barrier dysfunction

Abstract

Circulating components of neutrophil extracellular traps (NETs), especially histones, are associated with tissue injury during inflammatory conditions like sepsis. Commonly used as a NET biomarker, citrullinated histone 3 (H3Cit) may also functionally contribute to the NET-associated inflammatory response. To this end, we sought to examine the role of H3Cit in mediating microvascular endothelial barrier dysfunction. Here we show that H3Cit can directly contribute to inflammatory injury by disrupting the microvascular endothelial barrier. We found that endothelial responses to H3Cit are characterized by cell-cell adherens junction opening and cytoskeleton reorganization with increased F-actin stress fibers. Several signaling pathways often implicated in the transduction of hyperpermeability, such as Rho and MLCK, did not appear to play a major role; however, the adenylyl cyclase activator forskolin blocked the endothelial barrier effect of H3Cit. Taken together, the data suggest that H3Cit-induced endothelial barrier dysfunction may hold promise to treat inflammatory injury.

Keywords: Adherens junction; Cytoskeleton; H3Cit; Microcirculation; Permeability.

Figure 1.. H3Cit causes microvascular leakage of mouse mesenteric microvessels.

(A) Captured images during intravital microscopy of mice injected with FITC-albumin to observe microvascular leakage of mesenteric microvessels stimulated with H3Cit (10 μg/mL). (B) Quantification of transvascular flux (p<0.0001) using the equation IOI_Rel=(I_i-I_o)/I_i, where I_i=intensity inside the vessel and I_o=intensity outside the vessel. Data represented as mean +/− S.E.M. (n=4); * = p<0.05 vs Vehicle.

Figure 2.. H3Cit causes endothelial barrier dysfunction that is not dependent on cell death, Rho, or MLCK.

(A) Representative tracing of TER and (B) average maximum TER drop (p<0.0001) of HUVEC monolayers in response to increasing concentrations of H3Cit. Data represented as mean - S.E.M. (n=6–12); * = p<0.05 vs Vehicle. (C) LIVE/DEAD cell viability assay (absorbance at 530 nm of calcein AM to determine live cells and at 645 nm of ethidium homodimer-1 to determine dead cells; p<0.0001 ) [No tx = no treatment, (+) = positive control (70% methanol), Veh = vehicle control (0.1% BSA in PBS)]. (D-F) Average maximum TER drop of HUVEC monolayers in response to H3Cit (10 μg/mL) and Rho pathway inhibition by (D) Y27632 (10 μM, 30 min pre-treatment; p<0.0001) or (E) Rhosin (30 μM, 30 min pre-treatment; p<0.0001), or (F) MLCK pathway inhibition by MLCK inhibitor peptide 18 (10 μM, 30 min pre-treatment; p<0.0001). Data represented as mean - S.E.M. (n=8–12); * = p<0.05 vs Vehicle; ns = not significant.

Figure 3.. H3Cit causes endothelial barrier dysfunction by opening cell-cell adherens junctions and reorganizing the actin cytoskeleton.

(A) Immunofluorescence images and (B) intensity quantification of adherens junction protein VE-cadherin (green) at cell borders in HUVECs stimulated with H3Cit (10 μg/mL) for 1 minute (p=0.0026). Arrows indicate thinned segments. (C) Immunofluorescence images of F-actin (white) and G-actin (green) in HUVECs stimulated with H3Cit (10 μg/mL) for 1 minute and (D) quantification of the ratio of F-actin to G-actin (p=0.0074), indicating actin polymerization. Data represented as mean + S.E.M. (n=3); * = p<0.05 vs Vehicle.

Figure 4.. H3Cit-mediated endothelial barrier dysfunction can be prevented by adenylyl cyclase activator forskolin.

(A) Representative tracing and (B) average maximum TER drop of HUVEC monolayers in response to H3Cit (10 μg/mL) and barrier enhancement with forskolin (10 μM, 30 min pre-treatment; p<0.0001). Data represented as mean - S.E.M. (n=6–8); * = p<0.05 vs Vehicle, # = p<0.05 vs H3Cit.