br Methods and materials br

Methods and materials
Results
Discussion Many small molecules have been individually explored to enhance endothelial barrier both in vitro and in vivo, including S1P, forskolin, cAMP analogs, ROCK inhibitors, statins, and imatinib [[10], [11], [12],25,26]. In the current study, we applied two different assays (Transwell and ECIS) to compare the effects of multiple small molecules on endothelial cell barrier function. Our data confirmed that Ang-1, S1P, forskolin, and the cAMP analog 8CPT-2Me-cAMP significantly decreased endothelial permeability in a transwell assay, and improved resistance in the ECIS assay. Interestingly, we observed that the cAMP analog 8CPT-2Me-cAMP had significantly longer (>60 h) effect as compared to other small molecules (<5 h). One of the reasons might be biological half-life: S1P is metabolized very fast and its half time is about 30 min [27]; while the effect of 8CPT-2Me-cAMP lasts at least 24 h [28]. Cyclic AMP, a second messenger downstream of a G-coupled receptor, has been extensively studied to improve endothelial integrity primarily through protein kinase A- (PKA) or Epac-dependent mechanisms [14,15]. Previous studies have demonstrated that treatment with PKA selective cAMP analog 6-Bnz-cAMP, or the specific activator for Epac 8CPT-2Me-cAMP, could attenuate the disruptive endothelial permeability induced by thrombin [29]. In this current study, we observed no endothelial barrier improvement after treatment with the PKA selective cAMP analog (6-Bnz-cAMP), whereas the Epac-selective cAMP analog (8CPT-2Me-cAMP) significantly improved endothelial barrier. This discrepancy may be due to differing experimental systems: we measured the effect in a confluent endothelial monolayer, whereas Aslam [11] measured their effects in thrombin-compromised endothelial layers. The Epac agonist seems to improve endothelial barrier function through Gallein australia re-distribution. A recent study demonstrates that cAMP-Epac-Rap1-activated cells display increased circumferential actin bundle formation, and subsequent linkage between actin bundles and VE-cadherin through α- and β-catenins [30]. Similarly, our current study demonstrated a substantial actin re-distribution after treatment with 8CPT-2Me-cAMP. Further blockage of actin polymerization confirmed that the Epac agonist enhanced endothelial barrier primarily through actin re-organization. One of the challenges for lung and other whole-organ tissue engineering has been obtaining sufficient and functional autologous mature endothelium to repopulate large surface areas of organ microvasculature. The iPSC-derived ECFCs are an attractive option for autologous seeding of decellularized lung scaffold, since they exhibit surface marker profiles (i.e. CD31, 144, eNOS, and KDR) and cytoplasmic vWF production similar to mature ECs. Importantly, prior studies have shown that >108 ECFCs can be produced from each starting pluripotent stem cell: therefore clinically-relevant numbers of autologous endothelial cells may be obtained from such a source [17,18]. Furthermore, our data showed that iPSC-ECFCs could form a tighter cell barrier as compared to HUVECs. Intriguingly, after cultured in the lung scaffold for 5 days, iPSC-ECFCs displayed no sign of lung matrix breakdown (Supplemental Fig. 8) de-differentiation and the venous outflow increased overtime (Supplemental Fig. 9), suggesting some spontaneous formation of vascular barrier. qRT-PCR and western blot indicated an increase for most endothelial markers (e.g. PECAM1, CDH5, and KDR), as well as angiogenic and matrix remodeling markers (e.g. TEK, ANGPT1, NOTCH1, MMP14, and COL1A1), suggesting that iPSC-ECFCs are induced to a highly pro-angiogenic state when cultured on the organ matrix [31,32]. Endothelial cells that are actively angiogenic produce a number of factors involved in matrix remodeling (e.g. matrix metalloproteinases (MMPs), vascular endothelial growth factor (VEGF), fibroblast-derived growth factor (FGF)) to promote cell migration. Matrix remodeling can potentially damage the architecture of an organ scaffold, and may compromise basement membrane integrity and vascular barrier [[33], [34], [35]]. Thus, further work will be required to induce endothelial quiescence within the organ scaffold.