Hypoxia/reperfusion predisposes to atherosclerosis.

Surgical interventions on blood vessels bear a risk for intimal hyperplasia and atherosclerosis as a consequence of injury. A specific feature of intimal hyperplasia is the loss of vascular smooth muscle cell (VSMC) differentiation gene expression. We hypothesized that immediate responses following injury induce vascular remodeling. To differentiate injury due to trauma, reperfusion and pressure changes we analyzed vascular responses to carotid artery bypass grafting in mice compared to transient ligation. As a control, the carotid artery was surgically laid open only. In both, bypass or ligation models, the inflammatory responses were transient, peaking after 6h, whereas the loss of VSMC differentiation gene expression persisted. Extended time kinetics showed that transient carotid artery ligation was sufficient to induce a persistent VSMC phenotype change throughout 28 days. Transient arterial ligation in ApoE knockout mice resulted in atherosclerosis in the transiently ligated vascular segment but not on the not-ligated contralateral side. The VSMC phenotype change could not be prevented by anti-TNF antibodies, Sorafenib, Cytosporone B or N-acetylcysteine treatment. Surgical interventions involving hypoxia/reperfusion are sufficient to induce VSMC phenotype changes and vascular remodeling. In situations of a perturbed lipid metabolism this bears the risk to precipitate atherosclerosis.

Three-Dimensional Coculture Model to Analyze the Cross Talk Between Endothelial and Smooth Muscle Cells.

The response of blood vessels to physiological and pathological stimuli partly depends on the cross talk between endothelial cells (EC) lining the luminal side and smooth muscle cells (SMC) building the inner part of the vascular wall. Thus, the in vitro analysis of the pathophysiology of blood vessels requires coculture systems of EC and SMC. We have developed and validated a modified three-dimensional sandwich coculture (3D SW-CC) of EC and SMC using open µ-Slides with a thin glass bottom allowing direct imaging. The culture dish comprises an intermediate plate to minimize the meniscus resulting in homogenous cell distribution. Human umbilical artery SMC were sandwiched between coatings of rat tail collagen I. Following SMC quiescence, human umbilical vein EC were seeded on top of SMC and cultivated until confluence. By day 7, EC had formed a confluent monolayer and continuous vascular endothelial (VE)-cadherin-positive cell/cell contacts. Below, spindle-shaped SMC had formed parallel bundles and showed increased calponin expression compared to day 1. EC and SMC were interspaced by a matrix consisting of laminin, collagen IV, and perlecan. Basal messenger RNA (mRNA) expression levels of E-selectin, angiopoietin-1, calponin, and intercellular adhesion molecule 1 (ICAM-1) of the 3D SW-CC was comparable to that of a freshly isolated mouse inferior vena cava. Addition of tumor necrosis factor alpha (TNF α) to the 3D SW-CC induced E-selectin and ICAM-1 mRNA and protein induction, comparable to the EC and SMC monolayers. In contrast, the addition of activated platelets induced a significantly delayed but more pronounced activation in the 3D SW-CC compared to EC and SMC monolayers. Thus, this 3D SW-CC permits analyzing the cross talk between EC and SMC that mediate cellular quiescence as well as the response to complex activation signals.

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.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.