Role of the Lymphatics in Cardiac Disease.

Cardiovascular diseases remain the largest cause of death worldwide with recent evidence increasingly attributing the development and progression of these diseases to an exacerbated inflammatory response. As a result, significant research is now focused on modifying the immune environment to prevent the disease progression. This in turn has highlighted the lymphatic system in the pathophysiology of cardiovascular diseases owing, in part, to its established function in immune cell surveillance and trafficking. In this review, we highlight the role of the cardiac lymphatic system and its potential as an immunomodulatory therapeutic target in selected cardiovascular diseases.

Imaging the injured beating heart intravitally and the vasculoprotection afforded by haematopoietic stem cells.

AIMS: Adequate microcirculatory perfusion, and not just opening of occluded arteries, is critical to salvage heart tissue following myocardial infarction. However, the degree of microvascular perfusion taking place is not known, limited primarily by an inability to directly image coronary microcirculation in a beating heart in vivo. Haematopoietic stem/progenitor cells (HSPCs) offer a potential therapy but little is known about their homing dynamics at a cellular level and whether they protect coronary microvessels. This study used intravital microscopy to image the anaesthetized mouse beating heart microcirculation following stabilization. METHODS AND RESULTS: A 3D-printed stabilizer was attached to the ischaemia-reperfusion injured (IRI) beating heart. The kinetics of neutrophil, platelet and HSPC recruitment, as well as functional capillary density (FCD), was imaged post-reperfusion. Laser speckle contrast imaging (LSCI) was used for the first time to monitor ventricular blood flow in beating hearts. Sustained hyperaemic responses were measured throughout reperfusion, initially indicating adequate flow resumption. Intravital microscopy confirmed large vessel perfusion but demonstrated poor transmission of flow to downstream coronary microvessels. Significant neutrophil adhesion and microthrombus formation occurred within capillaries with the latter occluding them, resulting in patchy perfusion and reduced FCD. Interestingly, 'patrolling' neutrophils were also observed in capillaries. Haematopoietic stem/progenitor cells readily trafficked through the heart but local retention was poor. Despite this, remarkable anti-thromboinflammatory effects were observed, consequently improving microvascular perfusion. CONCLUSION: We present a novel approach for imaging multiple microcirculatory perturbations in the beating heart with LSCI assessment of blood flow. Despite deceptive hyperaemic responses, increased microcirculatory flow heterogeneity was seen, with non-perfused areas interspersed with perfused areas. Microthrombi, rather than neutrophils, appeared to be the major causative factor. We further applied this technique to demonstrate local stem cell presence is not a pre-requisite to confer vasculoprotection. This is the first detailed in vivo characterization of coronary microcirculatory responses post-reperfusion injury.

Tspan18 is a novel regulator of the Ca2+ channel Orai1 and von Willebrand factor release in endothelial cells.

Ca2+ entry via Orai1 store-operated Ca2+ channels in the plasma membrane is critical to cell function, and Orai1 loss causes severe immunodeficiency and developmental defects. The tetraspanins are a superfamily of transmembrane proteins that interact with specific 'partner proteins' and regulate their trafficking and clustering. The aim of this study was to functionally characterize tetraspanin Tspan18. We show that Tspan18 is expressed by endothelial cells at several-fold higher levels than most other cell types analyzed. Tspan18-knockdown primary human umbilical vein endothelial cells have 55-70% decreased Ca2+ mobilization upon stimulation with the inflammatory mediators thrombin or histamine, similar to Orai1-knockdown. Tspan18 interacts with Orai1, and Orai1 cell surface localization is reduced by 70% in Tspan18-knockdown endothelial cells. Tspan18 overexpression in lymphocyte model cell lines induces 20-fold activation of Ca2+ -responsive nuclear factor of activated T cell (NFAT) signaling, in an Orai1-dependent manner. Tspan18-knockout mice are viable. They lose on average 6-fold more blood in a tail-bleed assay. This is due to Tspan18 deficiency in non-hematopoietic cells, as assessed using chimeric mice. Tspan18-knockout mice have 60% reduced thrombus size in a deep vein thrombosis model, and 50% reduced platelet deposition in the microcirculation following myocardial ischemia-reperfusion injury. Histamine- or thrombin-induced von Willebrand factor release from endothelial cells is reduced by 90% following Tspan18-knockdown, and histamine-induced increase of plasma von Willebrand factor is reduced by 45% in Tspan18-knockout mice. These findings identify Tspan18 as a novel regulator of endothelial cell Orai1/Ca2+ signaling and von Willebrand factor release in response to inflammatory stimuli.