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Following a myocardial infarction (MI), the prognosis of patients is highly dependent upon the re-establishment of perfusion not only in the occluded coronary artery, but also within the coronary microcirculation. However, our fundamental understanding of the pathophysiology of the tiniest blood vessels of the heart is limited primarily because no current clinical imaging tools can directly visualise them. Moreover, in vivo experimental studies of the beating heart using intravital imaging have also been hampered due to obvious difficulties related to significant inherent contractile motion, movement of the heart brought about by nearby lungs and its location in an anatomically challenging position for microscopy. However, recent advances in microscopy techniques, and the development of fluorescent reporter mice and fluorescently conjugated antibodies allowing visualisation of vascular structures, thromboinflammatory cells and blood flow, have allowed us to overcome some of these challenges and increase our basic understanding of cardiac microvascular pathophysiology. In this review, the elegant attempts of the pioneers in intravital imaging of the beating heart will be discussed, which focussed on providing new insights into the anatomy and physiology of the healthy heart microvessels. The reviews end with the more recent studies that focussed on disease pathology and increasing our understanding of myocardial thromboinflammatory cell recruitment and flow disturbances, particularly in the setting of diseases such as MI.

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): British Heart Foundation Introduction Whilst blood flow restoration is critical following myocardial infarction (MI), ischemia-reperfusion injury (IRI) accounts for ~50% of the final infarct size. Elderly patients have a poorer prognosis which may be linked to increased coronary microvessels susceptibility to injury. The newly discovered and inflammatory cytokine, interleukin-36 (IL-36), could potentially mediate these disturbances. However, its role in myocardial IRI is not known. Aim This study firstly aimed to determine whether IL-36 and its receptor (IL-36R) were present in the heart, whether their expression varied in an injury and age-related manner, and whether topical application of its cytokines on the beating heart induces an inflammatory response. Secondly, we determined whether coronary microcirculatory disturbances, overall blood ventricular perfusion, and infarct size post-IRI were modified by age. Lastly, we investigated whether an IL-36 receptor antagonist (IL-36Ra) could confer vasculoprotection and reduce myocardial infarction. Methods Myocardial IRI was induced in adult (3-months) and aged (>18-months) mice. In some studies, recombinant mouse IL-36Ra (15ug/mouse) was injected intra-arterially. IL-36R/α/β, and VCAM-1 expression were investigated immunohistochemically or using western blots. Beating heart coronary microcirculation was imaged intravitally in vivo and also ex vivo using multiphoton microscopy. Topical cytokine application was also observed intravitally. Laser speckle contrast imaging was used to determine perfusion, and infarct size was measured using dual TTC/Evans Blue staining. Results IL-36R/α/β were expressed predominantly on the microvasculature of murine hearts, with some cardiomyocyte expression also observed. Expression of IL-36R/α/β and VCAM-1 significantly increased with injury and age (see Table). Topical application of all IL-36 cytokines induced a significant (p Conclusion These novel results are the first to demonstrate myocardial presence of IL-36 and its receptor. Our novel findings of enhanced coronary microcirculatory perturbations associated with age may explain the poorer outcomes in elderly MI patients. Importantly, we are the first to demonstrate that targeting IL-36 was vasculoprotective and may be a potential novel therapy for treatment of myocardial IRI.

Following myocardial infarction (MI), elderly patients have a poorer prognosis than younger patients, which may be linked to increased coronary microvessel susceptibility to injury. Interleukin-36 (IL-36), a newly discovered proinflammatory member of the IL-1 superfamily, may mediate this injury, but its role in the injured heart is currently not known. We first demonstrated the presence of IL-36(α/β) and its receptor (IL-36R) in ischemia/reperfusion-injured (IR-injured) mouse hearts and, interestingly, noted that expression of both increased with aging. An intravital model for imaging the adult and aged IR-injured beating heart in real time in vivo was used to demonstrate heightened basal and injury-induced neutrophil recruitment, and poorer blood flow, in the aged coronary microcirculation when compared with adult hearts. An IL-36R antagonist (IL-36Ra) decreased neutrophil recruitment, improved blood flow, and reduced infarct size in both adult and aged mice. This may be mechanistically explained by attenuated endothelial oxidative damage and VCAM-1 expression in IL-36Ra-treated mice. Our findings of an enhanced age-related coronary microcirculatory dysfunction in reperfused hearts may explain the poorer outcomes in elderly patients following MI. Since targeting the IL-36/IL-36R pathway was vasculoprotective in aged hearts, it may potentially be a therapy for treating MI in the elderly population.

Introduction Aspirin and a potent platelet P2Y12 inhibitor, such as prasugrel or ticagrelor, are not always sufficient to prevent thrombus formation in patients with ST-elevation MI (STEMI), leading to “slow flow” or “no reflow” effects after stenting. GPIIb/IIIa inhibitors, such as eptifibatide, may help in this setting, but are not used routinely due to their bleeding risk. GPVI has critical roles in thrombosis and a minimal role in haemostasis. Here we tested whether depletion of GPVI has effects on thrombus formation after MI in an animal model and investigated the effects of a novel platelet GPVI inhibitor, glenzocimab (a Fab fragment of a monoclonal antibody), on platelet activation and thrombus formation when combined with aspirin and ticagrelor. Methods We used intravital microscopy in a murine model of ST-elevation myocardial infarction and ischaemia-reperfusion injury to investigate microvascular thrombosis. We investigated the antithrombotic effects of adding glenzocimab (previously known as ACT017) to blood from healthy donors and 20 patients with ACS treated with aspirin and ticagrelor. We compared the effect of glenzocimab with the GPIIb/IIIa inhibitor eptifibatide ex-vivo. We stimulated platelets with collagen and atherosclerotic plaque material that was sourced from patients undergoing carotid endarterectomy. We investigated effects on platelet aggregation, spreading, signalling, adhesion, thrombin generation, thrombus formation and clot stability ex vivo. Results Genetic depletion of GPVI in an animal model of myocardial infarction reduced microvascular thrombosis. Ex vivo, aspirin and ticagrelor partially inhibited atherosclerotic plaque-induced platelet aggregation (assessed by multiple electrode aggregometry) by 48% compared to control (34±3 vs. 65±4 U; P Conclusions The addition of glenzocimab to aspirin and ticagrelor provides synergistic inhibition of multiple critical mechanisms of atherothrombosis. Glenzocimab and the GPIIb/IIIa inhibitor, eptifibatide, share many similar antithrombotic effects, although glenzocimab has less impact on mechanisms involved in haemostasis compared to eptifibatide. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Academy of Medical Sciences UK Clinical Lecturer Starter GrantRoyal Embassy of Saudi Arabia

Introduction Whilst blood flow restoration is critical following myocardial infarction (MI), ischemia-reperfusion injury (IRI) accounts for ~50% of the final infarct size. We have previously shown intravitally that myocardial IRI induces thromboinflammation and reduces functional capillary density (FCD) in adult mouse beating heart microcirculation in vivo. [1] The newly discovered and inflammatory cytokine, interleukin-36 (IL-36), could potentially mediate these disturbances. However, its role in myocardial IRI is not known. This study aimed to determine whether coronary microcirculatory disturbances and infarct size post-IRI were modified by age and gender. Secondly, we investigated if IL-36 (α/β) and its receptor (IL-36R) were present in the heart, and whether their expression varied in an injury and age-related manner. Lastly, we investigated whether an IL-36 receptor antagonist (IL-36Ra) could confer vasculoprotection and reduce myocardial infarction. Methods Myocardial IRI was induced in adult (3-months) and aged (>18-months) female mice, with gender differences assessed in adult male and female mice. Beating heart coronary microcirculation was imaged intravitally and also ex vivo using multiphoton microscopy. IL-36R/α/β, and VCAM-1 expression were investigated immunohistochemically or using western blots. In some studies, recombinant mouse IL-36Ra (15ug/mouse) was injected intra-arterially at 5 minutes pre-reperfusion and 60 minutes post-reperfusion. Infarct size was measured using dual TTC/Evans Blue staining. Results Significantly increased basal (p Conclusion Our novel findings of enhanced coronary microcirculatory perturbations associated with age may explain the poorer outcomes in elderly MI patients. Furthermore, the cellular nature of the thromboinflammatory response may explain the gender-related differences in outcome after MI. Importantly, we are the first to demonstrate that targeting IL-36 may be a potential novel therapy for treatment of myocardial IRI. Conflict of Interest No

Background Improving stem cell (SC) deformability using pre-treatment strategies, or isolating more deformable sub-populations, may prevent non-specific entrapment of injected cells, maintain circulating numbers and thus increase the likelihood of capture by microvessels in injured organs. However, nothing is currently known about the basic mechanical properties of SCs, particularly with regards their elastic characteristics. This study therefore aimed to determine the mechanical characteristics of haematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) with comparisons made to neutrophils. Methods Micromanipulation and atomic force microscopy (AFM) were used to quantitate mechanical properties following large and small deformations respectively of neutrophils, MSCs and naive and stromal cell-derived factor-1α (SDF-1ɑ) or hydrogen peroxide (H2O2) pre-treated HSCs. Results Neutrophils and HSCs underwent rupture at ∼80% deformation. Nominal rupture stress (σR), nominal rupture tension (TR) and the Young's/elastic modulus at large deformations was significantly higher for neutrophils indicating they were stiffer and less deformable than HSCs. Surprisingly, MSCs did not rupture and were as deformable as HSCs despite their large size. Pre-treatment increased HSC deformability as indicated by lower rupture force, σR, TR and Young's modulus at large deformations. AFM demonstrated that pre-treatment increased the Young's modulus at smaller deformations indicating the HSC surface stiffened. This was accompanied by increased F-actin accumulation and its localisation in the cell cortex. Conclusion This is the first study to precisely demonstrate that mechanical distinctions exist amongst different therapeutic SCs with regards their deformability and rupture response to applied stress. This can potentially be utilized as label-free markers in microfluidic cell sorting systems to separate sub-populations of potentially more therapeutic SCs.

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.

Concern regarding the quality of cold perfusion (QOP) during macroscopic assessment of procured kidneys is a common reason for discard. In the UK, QOP is routinely graded by both retrieving and implanting teams during back-bench surgery as: 1 (good), 2 (fair), 3 (poor) or 4 (patchy). We evaluated the association of this grading with organ utilization, graft outcomes, and agreement between teams. Data on all deceased-donor kidneys procured between January 2000 and December 2016 were analyzed for discard rates, while association with graft outcomes was studied in single adult transplants. Of 31,167 kidneys procured, 90.6%, 5.7%, 1.7%, and 2.1% were assigned grades 1, 2, 3, and 4, respectively, at retrieval. QOP was an independent risk factor of discard, with the highest rates observed in grade 3 kidneys (41.8%), compared to 6.5% in grade 1 (aOR 7.67, 95% CI 5.44-10.82, p < .001). Grading at retrieval was an independent predictor of delayed graft function (p = .019) and primary non-function (p = .001), but not long-term graft survival (p = .111). Implanting grade was an independent predictor of all three outcomes (p < .001, p < .001, and p = .002, respectively). Consistency of grading between teams was poor (Kappa = 0.179). QOP influences utilization and predicts outcomes, but a standardized and validated scoring system is required.

Treatment of myocardial infarction (MI) focuses on the rapid re-establishment of regional myocardial perfusion following a blockage in one or more of the coronary arteries. Although the culprit artery can be re-perfused, a significant proportion of patients still incur extensive myocardial damage resulting in heart failure. This is partly due to reperfusion paradoxically leading to additional tissue damage, a condition called ischaemia-reperfusion (IR) injury that has been ascribed to inadequate coronary microcirculatory perfusion. Nevertheless, the degree of microvascular perfusion is unknown, limited by an inability to clinically image coronary microvessels. Haematopoietic stem/progenitor cells (HSPCs) offer a potential vasculoprotective therapy but little is known about their homing dynamics and whether they do indeed protect coronary microvessels. We have recently reported that after systemic infusion, individual HSPCs were readily observed trafficking through the murine injured heart albeit with poor local retention. Despite this, remarkable anti-thrombo-inflammatory effects were observed, consequently improving microvascular perfusion as observed by intravital microscopic imaging, thus resulting in attenuated infarct size. This chapter assesses in-depth the coronary vasculoprotective potential of exogenous HSPCs and its assessment by imaging with primary focus on novel intravital microscopy technique applied to the beating heart.

Interactions between platelets, leukocytes and the vessel wall provide alternative pathological routes of thrombo-inflammatory leukocyte recruitment. We found that when platelets were activated by a range of agonists in whole blood, they shed platelet-derived extracellular vesicles which rapidly and preferentially bound to blood monocytes compared to other leukocytes. Platelet-derived extracellular vesicle binding to monocytes was initiated by P-selectin-dependent adhesion and was stabilised by binding of phosphatidylserine. These interactions resulted in the progressive transfer of the platelet adhesion receptor GPIbα to monocytes. GPIbα+-monocytes tethered and rolled on immobilised von Willebrand Factor or were recruited and activated on endothelial cells treated with TGF-β1 to induce the expression of von Willebrand Factor. In both models monocyte adhesion was ablated by a function-blocking antibody against GPIbα. Monocytes could also bind platelet-derived extracellular vesicle in mouse blood in vitro and in vivo Intratracheal instillations of diesel nanoparticles, to model chronic pulmonary inflammation, induced accumulation of GPIbα on circulating monocytes. In intravital experiments, GPIbα+-monocytes adhered to the microcirculation of the TGF-β1-stimulated cremaster muscle, while in the ApoE-/- model of atherosclerosis, GPIbα+-monocytes adhered to the carotid arteries. In trauma patients, monocytes bore platelet markers within 1 hour of injury, the levels of which correlated with severity of trauma and resulted in monocyte clearance from the circulation. Thus, we have defined a novel thrombo-inflammatory pathway in which platelet-derived extracellular vesicles transfer a platelet adhesion receptor to monocytes, allowing their recruitment in large and small blood vessels, and which is likely to be pathogenic. ispartof: HAEMATOLOGICA vol:105 issue:5 pages:1248-1261 ispartof: location:Italy status: published

A crucial step in enabling adoptive T cell therapy is the isolation of antigen (Ag)-specific CD8+ T lymphocytes. Mechanical changes that accompany CD8+ T lymphocyte activation and migration from circulating blood across endothelial cells into target tissue, may be used as parameters for microfluidic sorting of activated CD8+ T cells. CD8+ T cells were activated in vitro using anti-CD3 for a total of 4 days, and samples of cells were mechanically tested on day 0 prior to activation and on day 2 and 4 post-activation using a micromanipulation technique. The diameter of activated CD8+ T cells was significantly larger than resting cells suggesting that activation was accompanied by an increase in cell volume. While the Young's modulus value as determined by the force versus displacement data up to a nominal deformation of 10% decreased after activation, this may be due to the activation causing a weakening of the cell membrane and cytoskeleton. However, nominal rupture tension determined by compressing single cells to large deformations until rupture, decreased from day 0 to day 2, and then recovered on day 4 post-activation. This may be related to the mechanical properties of the cell nucleus. These novel data show unique biomechanical changes of activated CD8+ T cells which may be further exploited for the development of new microfluidic cell separation systems.

The ability to image biological tissues is critical to our understanding of a range of systems and processes. In the case of in situ living tissue, such imaging is hampered by the innate mechanical properties of the tissue. In many cases, this provides challenges in how to process large amounts of image data which may contain aberrations from movement. Generally, current tools require the provision of reference images and are unable to maintain temporal correlations within an image set. Here, we describe a tool-Tify-which can accurately predict a numerical quality score versus human scoring and can analyse image sets in a manner that allows the maintenance of temporal relationships. The tool uses regression-based techniques to link image statistics to image quality based on user provided scores from a sample of images. Scores calculated by the software correlate strongly with the scores provided by human users. We identified that, in most cases, the software requires users to score between 20-30 frames in order to be able to accurately calculate the remaining images. Importantly, our results suggest that the software can use coefficients generated from consolidated image sets to process images without the need for additional manual scoring. Finally, the tool is able to use a frame windowing technique to identify the highest quality frame from a moving window, thus retaining macro-chronological connections between frames. In summary, Tify is able to successfully predict the quality of images in an image set based on a small number of sample scores provided by end-users. This software has the potential to improve the effectiveness of biological imaging techniques where motion artefacts, even in the presence of stabilisation, pose a significant problem.

Haematopoietic stem and progenitor cell (HSC) therapy may be promising for the treatment of inflammatory bowel disorders (IBDs). However, clinical success remains poor, partly explained by limited HSC recruitment following systemic delivery. The mechanisms governing HSC adhesion within inflamed colon, and whether this event can be enhanced, are not known. An immortalised HSC-like line (HPC7) was pre-treated with hydrogen peroxide (H2O2), activated platelet releasate enriched supernatant (PES) or platelet microparticles (PMPs). Subsequent adhesion was monitored using adhesion assays or in vivo ischaemia–reperfusion (IR) and colitis injured mouse colon intravitally. Integrin clustering was determined confocally and cell morphology using scanning electron microscopy. Both injuries resulted in increased HPC7 adhesion within colonic mucosal microcirculation. H2O2 and PES significantly enhanced adhesion in vitro and in the colitis, but not IR injured, colon. PMPs had no effect on adhesion. PES and PMPs induced clustering of integrins on the HPC7 surface, but did not alter their expression. Adhesion to the colon is modulated by injury but only in colitis injury can this recruitment be enhanced. The enhanced adhesion induced by PES is likely through integrin distribution changes on the HPC7 surface. Improving local HSC presence in injured colon may result in better therapeutic efficacy for treatment of IBD.

We investigated the adhesive behavior of mesenchymal stem cells (MSC) in blood, which might influence their fate when infused as therapy. Isolated human bone marrow MSC (BMMSC) or umbilical cord MSC (UCMSC) adhered efficiently from flow to the matrix proteins, collagen, or fibronectin, but did not adhere to endothelial selectins. However, when suspended in blood, BMMSC no longer adhered to collagen, while UCMSC adhered along with many aggregated platelets. Neither MSC adhered to fibronectin from flowing blood, although the fibronectin surface did become coated with a platelet monolayer. UCMSC induced platelet aggregation in platelet rich plasma, and caused a marked drop in platelet count when mixed with whole human or mouse blood in vitro, or when infused into mice. In contrast, BMMSC did not activate platelets or induce changes in platelet count. Interestingly, isolated UCMSC and BMMSC both adhered to predeposited platelets. The differences in behavior in blood were attributable to expression of podoplanin (an activating ligand for the platelet receptor CLEC‐2), which was detected on UCMSC, but not BMMSC. Thus, platelets were activated when bound to UCMSC, but not BMMSC. Platelet aggregation by UCMSC was inhibited by recombinant soluble CLEC‐2, and UCMSC did not cause a reduction in platelet count when mixed with blood from mice deficient in CLEC‐2. We predict that both MSC would carry platelets in the blood, but their interaction with vascular endothelium would depend on podoplanin‐induced activation of the bound platelets. Such interactions with platelets might target MSC to damaged tissue, but could also be thrombotic. Stem Cells 2018;36:1062–1074

There is significant interest in the use of mesenchymal stem cells (MSCs) as a potential therapeutic modality in disease and disorder, particularly those with an inflammation-based component such as coronary, renal and hepatic diseases. While there is no question that MSCs possess the capability to manipulate an ongoing inflammatory injury, the recruitment of these cells to injured sites is generally poor, and thus, open to manipulation. Enhancing the localised recruitment of MSCs to injured tissues may enhance the efficiency and efficacy of this mode of therapy. A number of techniques exist in the literature to improve the recruitment of MSCs to injured tissues, including the use of cytokines, chemical modifications and coating with either synthetic or biological particles. In addition to enhancing MSC recruitment, there is an increasing body of work examining techniques which may enhance the anti-inflammatory activity of these cells. This review will summarise the literature around these topics. This first section of this review summarises the current literature with regard to MSC homing and their recruitment during conditions of injury. In relation to the anti-inflammatory activity of MSCs, the role of systemic versus local activity will be discussed. The second part of the review focuses on the role of pretreatments in MSC therapy and how these may have potential for not only enhancing the recruitment of MSCs, but also their anti-inflammatory capabilities. In summary, it is clear that there is significant potential to improve the efficiency of MSC therapy and the techniques discussed in this review may be central to this in the future.

To commemorate the auspicious occasion of the 30th anniversary of IPC, leading pioneers in the field of cardioprotection gathered in Barcelona in May 2016 to review and discuss the history of IPC, its evolution to IPost and RIC, myocardial reperfusion injury as a therapeutic target, and future targets and strategies for cardioprotection. This article provides an overview of the major topics discussed at this special meeting and underscores the huge importance and impact, the discovery of IPC has made in the field of cardiovascular research. DJH was funded by the British Heart Foundation (Grant Number FS/10/039/28270), the Rosetrees Trust, and is supported by the National Institute for Health Research University College London Hospitals Biomedical Research Centre. HEB was funded by the Danish Council for Independent Research (11-108354), the Danish Council for Strategic Research (11-115818), the Novo-Nordisk Foundation and Trygfonden. PF was funded by the Hungarian Scientific Research Fund (OTKA K 109737 and ANN 107803). HACF is funded by a Startup Grant of the ``Excellence Cluster Cardio-Pulmonary System'' (ECCPS) from the German Research Foundation (DFG, Bonn, Germany) and the ``Peter und Traudl Engelhorn-Stiftung'' (Weilheim, Germany) and in part by the Russian Government Program for competitive growth of Kazan Federal University, Kazan (Russian Federation). GH was supported by the German Research Foundation (He 1320/18-3 and SFB 1116/B8). TM was funded by the Japan Society for the Promotion of Science (Grant-in-Aid for Scientific Research \#23591086, \#2646113). MRS was funded by Novo-Nordisk Foundation. DG-D is supported by the Spanish Institute of Health, Instituto de Salud Carlos III (Grants PIE 13/00027, RETICS-RIC, RD12/0042/0021, and PI14/01431). RS and PF were funded by the European Foundation for the Study of Diabetes (EFSD) New Horizons Collaborative Research Initiative from the European Association for the Study of Diabetes (EASD) and by the European Cooperation in Science and Technology (COST EU-ROS). PF was funded by the Hungarian Scientific Research Fund (OTKA K 109737 and ANN 107803). Sí

Human bone marrow mesenchymal stem cells (hMSCs) have shown benefit in clinical trials of patients with liver disease. Efficient delivery of cells to target organs is critical to improving their effectiveness. This requires an understanding of the mechanisms governing cellular engraftment into the liver. Binding of hMSCs to normal/injured liver tissue, purified extracellular matrices, and human hepatic sinusoidal endothelial cells (HSECs) were quantified in static and flow conditions. To define the mechanisms underpinning hMSC interactions, neutralizing adhesion molecule antibodies were used. Fluorescently labelled hMSCs were infused intraportally into CCl4–injured mice with and without neutralizing antibodies. hMSCs expressed high levels of CD29/β1-integrin and CD44. Using liver tissue binding assays, hMSC adhesion was greatest in diseased human liver versus normal liver (32.2 cells/field versus 20.5 cells/field [P = 0.048]). Neutralizing antibodies against CD29 and CD44 reduced hMSC binding to diseased liver by 34% and 35%, respectively (P = 0.05). hMSCs rolled at 528 μm/second on HSECs in flow assays. This rolling was abolished by CD29 blockade on hMSCs and vascular cell adhesion molecule-1 (VCAM-1) blockade on HSECs. Firm adhesion to HSECs was reduced by CD29 (55% [P = 0.002]) and CD44 (51% [P = 0.04]) blockade. Neutralizing antibodies to CD29 and CD44 reduced hepatic engraftment of hMSCs in murine liver from 4.45 cells/field to 2.88 cells/field (P = 0.025) and 2.35 cells/field (P = 0.03), respectively. hMSCs expressed modest levels of chemokine receptors including CCR4, CCR5, and CXCR3, but these made little contribution to hMSC adhesion in this setting. Conclusion: hMSCs bind preferentially to injured liver. Rolling of hMSCs is regulated by CD29/VCAM-1, whereas CD29/CD44 interactions with VCAM-1, fibronectin, and hyaluronan on HSECs determine firm adhesion both in vitro and in vivo as demonstrated using a murine model of liver injury. (HEPATOLOGY 2012;56:1063–1073)

The use of stem cells is considered a promising therapy for tissue regeneration and repair, particularly for tissues injured through degeneration, ischemia and inflammation. Bone marrow (BM)-derived haematopoietic stem cells (HSCs) are rare populations of multipotent stem cells that have been identified as promising potential candidates for treating a broad range of conditions. Although research into the use of stem cells for regenerative medicine is on a steep upward slope, clinical success has not been as forthcoming. This has been primarily attributed to a lack of information on the basic biology of stem cells, which remains insufficient to justify clinical studies. In particular, while our knowledge on the molecular adhesive mechanisms and local environmental factors governing stem cell homing to BM is detailed, our understanding of the mechanisms utilized at injured sites is very limited. For instance, it is unclear whether mechanisms used at injured sites are location specific or whether this recruitment can be modulated for therapeutic purposes. In addition, it has recently been suggested that platelets may play an important role in stem cell recruitment to sites of injury. A better understanding of the mechanisms used by stem cells during tissue homing would allow us to develop strategies to improve recruitment of these rare cells. This review will focus on the status of our current understanding of stem cell homing to injured tissues, the role of platelets and directions for the future.

Aims Cells of the monocyte lineage are the most abundant inflammatory cells found in atherosclerotic lesions. Dominance of the inflammatory infiltrate by monocytes indicates that there is a disease-driven mechanism supporting their selective recruitment. Previous studies have demonstrated that interactions between endothelial cells (ECs) and platelets may promote monocyte recruitment. In this study, we sought to expand on this knowledge using a complex coculture model of the diseased vessel wall. Methods and results Using primary human cells in an in vitro flow-based adhesion assay, we found that secretory arterial smooth muscle cells (SMCs), cocultured with ECs, promote preferential recruitment of monocytes from blood in a TGF-β1-dependent manner. Approximately 85% of leucocytes recruited to the endothelium were CD14+. Formation of adhesive platelet bridges on ECs was essential for monocyte recruitment as platelet removal or inhibition of adhesion to the ECs abolished monocyte recruitment. Monocytes were recruited from flow by platelet P-selectin and activated by EC-derived CC chemokine ligand 2 (CCL2), although the presentation of CCL2 to adherent monocytes was dependent upon platelet activation and release of CXC chemokine ligand 4 (CXCL4). In an intravital model of TGF-β1-driven vascular inflammation in mice, platelets were also necessary for efficient leucocyte recruitment to vessels of the microcirculation in the cremaster muscle. Conclusions In this study, we have demonstrated that stromal cells found within the diseased artery wall may promote the preferential recruitment of monocytes and this is achieved by establishing a cascade of interactions between SMCs, ECs, platelets, and monocytes.

Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These were investigated in vivo following hepatic ischaemia-reperfusion (IR) injury and in vitro using flow-based adhesion assays.Partial IR was induced in anaesthetised WT or PECAM-1(-/-) mice for 90 min. Recruitment of systemically administered HSCs was monitored and effects of function blocking antibodies against alpha(4)beta(1) integrin, CD18, CD44, PECAM-1 or VCAM-1 investigated. The kinetics and molecular events governing adhesion to murine cardiac endothelial cells in vitro were also determined. Effects of conditioned media from IR injured liver on HSC adhesion molecule expression was determined by FACS.Administered HSCs homed predominantly to lungs rather than liver, highlighting a potential therapeutic hurdle. Hepatic HSC recruitment following IR injury was inhibited by anti-alpha(4)beta(1) and anti-VCAM-1 antibodies. A role for alpha(4)beta(1) was also confirmed using flow-based adhesion assays. Incubating HSCs with conditioned media from IR injured liver increased alpha(4)beta(1) expression. CD18, CD44 and PECAM-1 were not involved in recruitment.This novel study demonstrates that the alpha(4)beta(1)/VCAM-1 pathway mediates HSC recruitment to injured liver. Manipulating this pathway may enhance delivery of HSCs to the liver.

The role of collagen receptor complex GPVI-FcR gamma-chain, PLCgamma2 and LAT in laser-induced thrombosis is unclear. Controversy surrounds whether collagen is exposed in this model or whether thrombosis is dependent on thrombin. This study hypothesized that collagen exposure plays a critical role in thrombus formation in this model, which was tested by investigating contributions of FcR gamma-chain, LAT, PLCgamma2 and thrombin.Thrombi were monitored using intravital microscopy in anesthetized wild-type and FcR gamma-chain, LAT and PLCgamma2 knockout mice. Hirudin (thrombin inhibitor) was administered to wild-type and FcR gamma-chain knockout mice.Significantly reduced thrombus formation was observed in FcR gamma-chain and PLCgamma2 knockouts with a greater decrease observed in LAT knockouts. Dramatic reduction was observed in wild-types treated with hirudin, with abolished thrombus formation only observed in FcR gamma-chain knockouts treated with hirudin.GPVI-FcR gamma-chain, LAT and PLCgamma2 are essential for thrombus generation and stability in this laser-induced model of injury. More importantly, a greater role for LAT was identified, which may reflect a role for it downstream of a second matrix protein receptor. However, inhibition of platelet activation by matrix proteins and thrombin generation are both required to maximally prevent thrombus formation.

Background/Aim:Helicobacter pylori is associated with an increased risk of peptic ulcer disease development and recurrence. Ulcer healing is dependent upon angiogenesis, requiring endothelial cell (EC) proliferation and chemotaxis. This study determined whether extracts of H. pylori affected EC proliferation and/or chemotaxis in vitro. Methods: The effects of water and broth extracts of three genotypically different strains of H. pylori and of single strains of Campylobacter jejuni and Escherichia coli on human dermal microvascular EC (HuDMEC) and human umbilical vein EC (HUVEC) were assessed. Tetrazolium dye (MTT) proliferation, dual staining cell viability, flow cytometry, and microchemotaxis assays were performed. Results:H. pylori (all strains) and C. jejuni inhibited HuDMEC (p < 0.01) and HUVEC (p < 0.01) proliferation and decreased the proportion of HUVECs in the S phase of the cell cycle. E. coli had no effect on EC proliferation. The levels of vascular endothelial growth factor stimulated chemotaxis were significantly greater (p < 0.01) than the levels of basal migration for both control and extract-treated ECs. However, none of the bacterial extracts affected EC basal migration or chemotaxis. Conclusion:H. pylori extracts inhibit HuDMEC and HUVEC proliferation in vitro by a cytostatic, strain-independent mechanism. A similar antiproliferative effect of C. jejuni was observed. Our findings suggest that both H. pylori and C. jejuni have the ability to inhibit one of the key stages of angiogenesis which may have implications in peptic ulcer disease.

Background The clinical success of stem cell (SC) therapy for myocardial infarction is compromised by poor cardiac homing following systemic delivery. As such therapy may depend on beneficial paracrine effects, it is a further hindrance that little is known about the inflammatory response dynamics within myocardial microcirculation in vivo. Methods 3D-printed stabilisers were bonded to the beating heart of anaesthetised (ketamine/xylazine) mice to enable confocal intravital imaging of ventricular microcirculation. PE-anti-Gr-1 and APC-anti-CD41 antibodies were injected to label neutrophils and platelets respectively with FITC-BSA enabling blood flow visualisation. In some mice, haematopoietic SCs (HSCs; HPC-7s) were introduced intra-arterially. IR injury was induced by 45 min (reversible) ligation of the LAD artery. Results Neutrophil adhesion and platelet accumulation were both significantly (p Discussion Intravital microscopy has allowed successful visualisation of the microvascular inflammatory response and HSC homing events in the beating mouse heart post-reperfusion. Subsequent experiments will allow us to assess whether HSC administration can confer vasculoprotection.

During an inflammatory response, lymphocyte recruitment into tissue must be tightly controlled because dysregulated trafficking contributes to the pathogenesis of chronic disease. Here we show that during inflammation and in response to adiponectin, B cells tonically inhibit T cell trafficking by secreting a peptide (PEPITEM) proteolytically derived from 14.3.3 zeta delta (14.3.3.ζδ) protein. PEPITEM binds cadherin-15 on endothelial cells, promoting synthesis and release of sphingosine-1 phosphate, which inhibits trafficking of T cells without affecting recruitment of other leukocytes. Expression of adiponectin receptors on B cells and adiponectin-induced PEPITEM secretion wanes with age, implying immune senescence of the pathway. Additionally, these changes are evident in individuals with type 1 diabetes or rheumatoid arthritis, and circulating PEPITEM in patient serum is reduced compared to that of healthy age-matched donors. In both diseases, tonic inhibition of T cell trafficking across inflamed endothelium is lost. Control of patient T cell trafficking is re-established by treatment with exogenous PEPITEM. Moreover, in animal models of peritonitis, hepatic ischemia-reperfusion injury, Salmonella infection, uveitis and Sjogren's syndrome, PEPITEM reduced T cell recruitment into inflamed tissues.

55 Establishing a model for intravital imaging of the beating murine heart {#article-title-2} Introduction: While stem cell (SC)-based therapy following heart injury holds promise, clinical success is compromised by poor SC recruitment to the heart following infusion. One solution is to identify strategies that may increase SC capture in cardiac microvessels. We have routinely used intravital microscopy (IVM), a method of live in vivo imaging of the microcirculation, to monitor cell trafficking. To do so in the heart would help facilitate research aimed at improving SC delivery. However, IVM of the beating mouse heart remains challenging due to difficulties with organ access and impractical degrees of tissue motion. The study presents a method that allows for cardiac IVM using a 3D-printed stabilizing device. Methods: Anaesthetized mice were artificially ventilated using a small rodent ventilator which delivered medical oxygen. PEEP was applied by immersing the ventilator outlet in 4cm of ddH2O. A chest wall incision, expanded with retractors, facilitated the adhesive positioning of a specially designed tissue stabilizer onto the heart surface. This limited respiratory and cardiac contractile motion in a small area without compromising overall heart function. Imaging was performed through a small hole in the centre of the stabilizer. Preliminary studies were performed in the myocardial ischemia-reperfusion injury model. This injury was established by occlusion of the left anterior descending artery (LAD). The vessel was occluded for 45 minutes using a small piece of plastic tubing within a loop which was placed under the LAD. Identification of vascular dynamics (e.g. cell recruitment, monitoring of vascular no-reflow) took place at various points during reperfusion. Administration of FITC-BSA allowed for visualisation of the microvasculature. Endogenous neutrophils were labelled by intra-arterial administration of 6µg eFluor-660 labelled anti-Gr-1 antibody. Results: Infusion of FITC-BSA permitted visualization of the coronary microcirculation. Vascular integrity was not disturbed and flow was visible as a result of application of the stabiliser. Endogenous (labelled) neutrophils were easily identified circulating in the cardiac microcirculation. Following cardiac IR, we identified significantly enhanced neutrophil recruitment as early as 60 minutes post-reperfusion, when compared to sham-operated control animals. Furthermore, using FITC-BSA visualization of the coronary microcirculation, we identified areas of vascular no-reflow following cardiac IR. Finally, systemically administered SCs (fluorescently labelled HSCs) could be identified trafficking through the cardiac microvasculature following cardiac IR injury. Conclusion: We present a model for intravital imaging of the mouse beating heart in vivo. This method may be useful in monitoring cell dynamics and microvascular disturbances in the heart at the single cell level. # 56 Altered RyR microdomains lead to more Ca2+ waves in non-coupled RyRs after myocardial infarction {#article-title-3} Purpose: In ventricular myocytes of large mammals, a significant number of ryanodine receptors (RyRs) are not in couplons (i.e. non-coupled RyRs). Previous research from our lab has shown that these non-coupled receptors lack CaMKII and NOX2 microdomain modulation. Furthermore, after MI TTs are lost and hence the fraction of non-coupled RyRs is increased. We investigated whether this affected microdomain signaling and diastolic Ca2+ waves which are potentially arrhythmogenic. Methods: Using an established pig model of chronic ischemia and myocardial infarction (MI, N=16), we studied myocytes from the area adjacent to the MI and compared these to myocytes from SHAM pigs (N=13) using whole-cell voltage clamp with Fluo-4 as a [Ca2+]i indicator together with confocal line scan imaging. Myocytes were stimulated at a high frequency (2 Hz) in the presence of isoproterenol (10 nM; ISO). Ca2+ waves were recorded during a 15 s rest period following stimulation and assigned to different subcellular regions categorized as coupled or non-coupled RyRs using a specific algorithm. Results: Conditioning myocytes at high frequency in the presence of ISO induced more Ca2+ waves in MI myocytes (0.21 ± 0.1 #waves/100µm/s; n=62) compared to SHAM myocytes (0.15 ± 0.1 #waves/100µm/s; n=59). No differences were found in wave foci, but a significant reduction in foci recruitment occurred after MI (57 % in MI vs. 70 % in SHAM). Moreover, in healthy myocytes more Ca2+ waves occurred in coupled compared to non-coupled in a 2:1 ratio. In MI myocytes, however, the subcellular site of wave occurrence was reversed to non-coupled sites resulting in a 1:2 ratio for coupled:non-coupled RyRs. Specific blockers directly (S107) or indirectly (AIP inhibiting CaMKII, mitoTEMPO scavenging mitochondrial ROS) targeting RyRs were investigated. In healthy myocytes, none of the blockers significantly affected wave frequency, neither at the global nor in the coupled versus non-coupled RyRs. However, in myocytes from MI animals, S107, mitoTEMPO and AIP all reduced the global increase in wave frequency. Subcellular analysis of different RyR subsets showed that in MI S107 (0.06 ± 0.1 #waves/100µm/s; n=27), AIP (0.06 ± 0.1 #waves/100µm/s; n=25) and mitoTEMPO (0.08 ± 0.1 #waves/100µm/s; n=22) reduced the wave frequency in non-coupled RyRs without significantly affecting coupled RyRs. Conclusion: After MI, Ca2+ waves arise predominantly in non-coupled RyRs. The non-coupled RyRs after MI are modulated via local microdomains of CaMKII and mitochondrial ROS. These data highlight the arrhythmogenic nature of the non-coupled RyR clusters, which are at a higher density after MI, and suggest there may be a potential for selective pharmacological targeting. # 57 Intravital microscopy (IVM), a new method for in vivo imaging of monocyte homing in a mouse hind limb arteriogenesis model {#article-title-4} Therapeutic augmentation of collateral vessel growth (arteriogenesis) is of particular clinical interest. Monocytes are key players within this process. Quantification of collateralization in small animal models is, however, difficult and the commonly used techniques are post-mortem or histological assays. Live imaging of monocyte homing to sites of arteriogenesis in vivo has not been demonstrated until now. Therefore, an intravital microscopy (IVM) technique was established to visualize this process. The IVM is a non-invasive optical imaging technique offering high resolution, deep tissue penetration and video capturing. Nowadays only already located (homed) cells could be detected by post mortem examination of the tissue. Real time transmigration and interaction of cells had never been visualized under these circumstances. By the use of this innovative and unique method we were able to examine the kinetics of monocyte homing in vivo and endogenous development of collateral arteries within the hind limb of C57BL6 mice after ligature of one femoral artery. The morphology of the collateral vessels in the adductor muscle was determined by 3D imaging and blood flow was visualized within the growing arteries. Our results were validated by histological workup and laser Doppler perfusion imaging (LDPI). For the in vivo experiments, mice were anesthetized intraperitoneally and placed on a heating plate (37°C). Then, the leg was fixed between two adjustable stamps and care was taken to keep the region of interest moist. A cover glass was positioned on top of the stamps and adjusted before imaging was started. A Zeiss LSM 710 NLO with a 20x water immersion lens (NA 1.0) or a Leica SP 5 LSM microscope with a 10x dry lens (NA 0.4) were used in either 2 Photon or LSM mode, respectively. With both systems we were able to achieve deep tissue penetration up to 1 mm, thereby capturing multi-colour videos of living cells. Histological workup of hind limb tissue sections of C57BL6 mice approved the increase of collateralization. The ratio of collateral arteries (ligated/unligated leg) was calculated (each group n=4). The intra individual ratio increased from 1.0±0.07 (prior to ligation) to 1.32±0.17 two weeks and 1.44±0.11 three weeks after ligation (P

Hematopoietic stem cells (HSCs) migrate to injury sites and aid in tissue repair. However, clinical success is poor and is partially due to limited HSC recruitment. We hypothesized that HSC pretreatment with H2O2 would enhance their recruitment to injured gut. As HSCs are rare cells, the number of primary cells obtained from donors is often inadequate for functional experiments. To circumvent this, in this study we utilized a functionally relevant cell line, HPC-7. Anesthetized mice were subjected to intestinal ischemia-reperfusion (IR) injury, and HPC-7 recruitment was examined intravitally. Adhesion to endothelial cells (ECs), injured gut sections, and ICAM-1/VCAM-1 protein were also quantitated in vitro. H2O2 pretreatment significantly enhanced HPC-7 recruitment to injured gut in vivo. A concomitant reduction in pulmonary adhesion was also observed. Enhanced adhesion was also observed in all in vitro models. Increased clustering of α4 and β2 integrins, F-actin polymerization, and filopodia formation were observed in pretreated HPC-7s. Importantly, H2O2 did not reduce HPC-7 viability or proliferative ability. HPC-7 recruitment to injured gut can be modulated by H2O2 pretreatment. This may be through increasing the affinity or avidity of surface integrins that mediate HPC-7 homing to injured sites or through stimulating the migratory apparatus. Strategies that enhance hematopoietic stem/progenitor cell recruitment may ultimately affect their therapeutic efficacy.

IL-17 secreting CD4 (Th17) and CD8 (Tc17) T cells have been implicated in immune-mediated liver diseases, but the molecular basis for their recruitment and positioning within the liver is unknown.The phenotype and migratory behaviour of human liver-derived Th17 and Tc17 cells were investigated by flow cytometry and chemotaxis and flow-based adhesion assays. The recruitment of murine Th17 cells to the liver was studied in vivo using intra-vital microscopy.IL-17(+) T cells comprised 1-3% of the T cell infiltrate in inflammatory liver diseases and included both CD4 (Th17) and CD8 (Tc17) cells. They expressed RORC and the IL-23 receptor and included subsets that secreted IL-22 and interferon-γ. Th17 and Tc17 cells expressed high levels of CXCR3 and CCR6, Tc17 cells also expressed CXCR6. Binding to human sinusoidal endothelium from flow was dependent on β1 and β2 integrins, CXCR3, and, in the case of Th17 cells, VAP-1. Th17 recruitment via sinusoids in mice with liver inflammation was reduced by treatment with antibodies against CXCR3 ligands, confirming the role of CXCR3 in Th17 recruitment in vivo. In human liver, IL-17(+) cells were detected in portal infiltrates close to inflamed bile ducts expressing the CCR6 ligand CCL20. Cytokine-treated human cholangiocytes secreted CCL20 and induced CCR6-dependent migration of Th17 cells suggesting that local cholangiocyte chemokine secretion localises Th17 cells to bile ducts.CXCR3 promotes recruitment of Th17 cells from the blood into the liver in both human and murine liver injury. Their subsequent positioning near bile ducts is dependent on cholangiocyte-secreted CCL20.

Tetraspanin CD151 is associated with laminin-binding integrins and controls tumor cell migration and invasion. By analyzing responses of breast cancer cells to various growth factors, we showed that depletion of CD151 specifically attenuates transforming growth factor β1 (TGFβ1)–induced scattering and proliferation of breast cancer cells in three-dimensional Matrigel. CD151-dependent cell scattering requires its association with either α3β1 or α6 integrins, but it is independent of the recruitment of CD151 to tetraspanin-enriched microdomains. We also found that CD151 regulates the compartmentalization of TGF-β type I receptor (TβRI/ALK-5) and specifically controls the TGFβ1-induced activation of p38. In contrast, signaling leading to activation of Smad2/3, c-Akt, and Erk1/2 proteins was comparable in CD151(+) and CD151(−) cells. Attenuation of TGFβ1-induced responses correlated with reduced retention in the lung vascular bed, inhibition of pneumocyte-induced scattering of breast cancer cells in three-dimensional Matrigel, and decrease in experimental metastasis to the lungs. These results identify CD151 as a positive regulator of TGFβ1-initiated signaling and highlight the important role played by this tetraspanin in TGFβ1-induced breast cancer metastasis. Cancer Res; 70(14); 6059–70. ©2010 AACR.