It is well established that a host of factors within

It is well established that a host of factors within the injured microenvironment are capable of activating circulating Deferoxamine and platelets and thus permitting their endothelial adhesion during inflammatory processes (Mori et al., 2005a; Vowinkel et al., 2007; Sprague and Khalil, 2009; Santen et al., 2010). A potent leukocyte pro-adhesive modulator found within many inflammatory environments, particularly the ischaemic and colitis gut, is the free radical hydrogen peroxide (H2O2), generated locally by endothelium and infiltrating neutrophils (Fraticelli et al., 1996; Damiani et al., 2007). Similar factors most likely activate trafficking SCs as they circulate through damaged tissue. We hypothesised that pre-treatment of HSCs with such factors would increase the likelihood of their adhesion. Indeed, we have previously demonstrated that H2O2 pre-treatment enhances adhesion of an immortalised HSC-like line (HPC7) within the ischaemia-reperfusion (IR) injured small intestine (SI) in vivo (Kavanagh et al., 2013a). This increase was mediated by clustering α4 and β2 integrins on the HPC7 surface, increasing their affinity for endothelial counterligands such as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and inducing HPC7 pro-migratory filopodia formation. Whether H2O2 pre-treated HSCs can adhere within the injured colon more efficiently than naïve HSCs is not known. An alternative strategy could involve HSC pre-treatment with the use of biological factors such as platelet derived microparticles (PMPs). On activation, platelets release these small (<1μm) membranous vesicles that express many of the surface receptors found on platelets (Burnouf et al., 2014). PMPs are the biggest source of microparticles within humans and are known to be increased in various pathologies, including Crohn\'s disease (Chamouard et al., 2005). Initially, it was thought that PMPs were cellular debris without any distinct physiological or pathological function. However, it is increasingly evident that PMPs have many roles, including an ability to increase neutrophil adhesion by transfer of adhesion molecules to their surface (Jy et al., 1995). The use of PMPs as a pre-treatment option for HSCs is attractive as they have not only been shown to enhance HSC recruitment to bone marrow (Janowska-Wieczorek et al., 2001), but also increase cellular repair efficiency independently of affects on adhesion (Burnouf et al., 2014; Mause et al., 2010). Determining whether systemic administration of transplanted HSCs can efficiently deliver cells to intestinal tissues, and whether this phenomenon can be enhanced, has received little attention, with no studies conducted in the injured colon in vivo. Therefore in this study, we used intravital microscopy, a methodology with single-cell sensitivity, to firstly detail the homing kinetics of an immortalised HSC-like line (HPC7) to the murine colon following two distinctly different injuries, namely an acute IR injury and a more chronic colitis injury. We further elucidated the molecular adhesive mechanisms governing HSC homing to injured colonic microvessels. Having already demonstrated an ability for H2O2 to improve HPC7 retention within IR injured SI, we tested this pre-treatment strategy within the two colon injury models. The efficacy of this chemical strategy was also compared with HPC7s pre-coated with PMPs. This is the first study to have tested HSC homing to two models of colonic injury and also utilised two distinctly different pre-treatment strategies in an attempt to enhance their local presence.
Materials and methods
Statistics All statistical analyses and graph plotting were performed using Prism GraphPad v4 (GraphPad Software Inc., USA). The area under the curve (AUC) values were calculated using the same software for each intravital and laser speckle experiment from the serial data plots and presented alongside the serial plots as bar charts. This is the most appropriate way of statistically interpreting serial measurements such as these. The AUC mean+SEM values were calculated and ANOVA with Dunnett\'s post-test used to test for significant differences. Some experiments were analysed using Student\'s t-test as appropriate. Details of statistical tests are contained within figure legends. Data is presented as mean+standard error or mean (SEM) in all cases.