Evidence That Cingulin Regulates Endothelial Barrier Function In Vitro and In Vivo

Objective— Cingulin is a cytoplasmic component of tight junctions. Although modulation of cingulin levels in cultured epithelial model systems has no significant effect on barrier function, evidence from cingulin knockout mice suggests that cingulin may be involved in the regulation of the behavior of epithelial or endothelial cells. Here, we investigate the role of cingulin in the barrier function of endothelial cells. Approach and Results— We show that cingulin is expressed in human endothelial cells of the skin, brain, and lung in vivo and in vitro. Endothelial cingulin colocalizes and coimmunoprecipitates with the tight junction proteins zonula occludens-1 and guanine nucleotide exchange factor-H1. Cingulin overexpression in human umbilical vein endothelial cell induces tight junction formation, increases transendothelial electric resistance, and strengthens barrier function for low and high molecular weight tracers. In contrast, cultured endothelial cells lacking cingulin are more permeable for low molecular weight tracers. In cingulin knockout mice, neurons of the area postrema and Purkinje cells show an increased uptake of small molecular weight tracers indicating decreased barrier function at these sites. Conclusions— We demonstrate that cingulin participates in the modulation of endothelial barrier function both in human cultured cells in vitro and in mouse brains in vivo. Understanding the role of cingulin in maintaining tight barriers in endothelia may allow developing new strategies for the treatment of vascular leak syndromes.</p

A Peptide to Reduce Pulmonary Edema in a Rat Model of Lung Transplantation

<div><p>Background</p><p>Despite significant advances in organ preservation, surgical techniques and perioperative care, primary graft dysfunction is a serious medical problem in transplantation medicine in general and a specific problem in patients undergoing lung transplantation. As a result, patients develop lung edema, causing reduced tissue oxygenation capacity, reduced lung compliance and increased requirements for mechanical ventilatory support. Yet, there is no effective strategy available to protect the grafted organ from stress reactions induced by ischemia/reperfusion and by the surgical procedure itself.</p><p>Methods</p><p>We assessed the effect of a cingulin-derived peptide, XIB13 or a random peptide in an established rat model of allogeneic lung transplantation. Donor lungs and recipients received therapeutic peptide at the time of transplantation and outcome was analyzed 100min and 28 days post grafting.</p><p>Results</p><p>XIB13 improved blood oxygenation and reduced vascular leak 100min post grafting. Even after 28 days, lung edema was significantly reduced by XIB13 and lungs had reduced fibrotic or necrotic zones. Moreover, the induction of an allogeneic T cell response was delayed indicating a reduced antigen exchange between the donor and the host.</p><p>Conclusions</p><p>In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.</p></div

Improved tissue morphology and reduced edema in XIB13-treated lung grafts in rats 28 days post LTX.

<p>(A) 28 days post LTX, lung morphology in histological sections was graded as necrotic, fibrotic or with maintained alveolar structures in hematoxylin & eosin-stained sections combined with CD31 stains to identify vascularity of grafts (scale bar 200μm). Mean percent values and SD of necrotic, fibrotic or vascularized lung areas are shown for 5 animals per treatment group (p = 0,215); (B) To quantify tissue edema, wet-to-dry ratios of lungs were determined 28 days post LTX (mean +/- SD, right lungs p = 0,010, left lungs p = 0,003; n = 9/group); (C) mRNA expression of MPO, IL-6, TNFα and IL-1β in lung tissue of study animals. Values were expressed as fold change relative to untransplanted left lungs from donor animals (mean +/- SD; n = 5/group; * denotes p≤0,05; n.s. denotes not significant; p≥0,15 for all other targets).</p

XIB13 has no effect on ventilation-induced lung injury in mice.

<p>(A) Tissue edema was quantified by determining Wet/Dry ratios of lungs (mean +/- SD, p = 0,059; n = 9/group); (B) cell influx was assessed in cytospin preparations of BALF (mean +/- SD, p = 0,337; n = 9/group); (C) cytokine levels (IL-6 and TNFα) in BALF were determined by ELISA. (mean +/- SD, p = 0,727 for IL-6, p = 0,190 for TNFα; n = 9/group).</p

XIB13-treated rats have reduced allo-reactive T cells.

<p>T cells from recipient rats retrieved 28 days post LTX were mixed with irradiated spleen cells from donors and showed a significant reduction in proliferation as compared to control-treated animals (A, mean +/- SD, p = 0,000; n = 6). (B) The peptide did not directly alter lymphocyte proliferation: PBMCs were incubated with XIB13 or control peptide for 3 days (mean +/- SD, p = 0,178; n = 3); (C) The effect was not due to increased exposure of recipients to donor cells as measured by numbers of donor DNA copies in the recipients blood (B, mean +/- SD, p = 0,360; n = 6). XIB13 had no effect on Wet/Dry-ratios, cell influx and cytokine secretion in ventilation-induced lung injury in mice not subjected to any surgical procedure (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0142115#pone.0142115.g004" target="_blank">Fig 4</a>).</p

Positive agglutination by indirect Coombs test to assess anti-donor antibodies from recipient serum.

<p>Positive agglutination by indirect Coombs test to assess anti-donor antibodies from recipient serum.</p