Keratinocytes play a critical role in maintaining epidermal barrier function. Activated protein C (APC), a natural anticoagulant with anti-inflammatory and endothelial barrier protective properties, significantly increased the barrier impedance of keratinocyte monolayers, measured by electric cell substrate impedance sensing and FITC-dextran flux. In response to APC, Tie2, a tyrosine kinase receptor, was rapidly activated within 30 min, and relocated to cell-cell contacts. APC also increased junction proteins zona occludens, claudin-1 and VE-cadherin. Inhibition of Tie2 by its peptide inhibitor or small interfering RNA abolished the barrier protective effect of APC. Interestingly, APC did not activate Tie2 through its major ligand, angiopoietin-1, but instead acted by binding to endothelial protein C receptor, cleaving protease-activated receptor-1 and transactivating EGF receptor. Furthermore, when activation of Akt, but not ERK, was inhibited, the barrier protective effect of APC on keratinocytes was abolished. Thus, APC activates Tie2, via a mechanism requiring, in sequential order, the receptors, endothelial protein C receptor, proteaseactivated receptor-1, and EGF receptor, which selectively enhances the PI3K/Akt signaling to enhance junctional complexes and reduce keratinocyte permeability.

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Objective. To investigate whether proteaseactivated receptor 1 (PAR-1) and/or PAR-2 promotes the invasiveness/proliferation of synovial fibroblasts (SFs) and to determine the signaling mechanisms of these pathways.Methods. SFs were isolated from the synovial tissue of patients with rheumatoid arthritis (RA), patients with osteoarthritis (OA), and PAR-1-or PAR-2-knockout (KO) mice. Expression of PAR-1 and PAR-2 was detected by immunofluorescence and Western blotting. The invasion and proliferation of SFs were measured by invasion assay and MTT assay, respectively. Matrix metalloproteinase 2 (MMP-2) and MMP-9 were detected by zymography, and cytokines were measured by enzyme-linked immunosorbent assay.Results. PAR-1 and PAR-2 were colocalized with SFs in RA and OA synovium and, to a considerably lesser extent, in normal synovium. Inhibition of PAR-2 by small interfering RNA (siRNA) inhibited RASF invasion and proliferation, whereas blocking of PAR-1 by siRNA had the reverse effects. SFs from PAR-2-KO mice exhibited slower rates of proliferation and invasion. SFs from PAR-1-KO mice produced less MMP-2 and, in response to tumor necrosis factor ␣ (TNF␣) stimulation, had increased MMP-9 secretion when compared to SFs from wild-type and PAR-2-KO mice. Inhibition of PAR-1, but not PAR-2, stimulated the secretion of interleukin-17 (IL-17) and TNF␣ by RASFs. Furthermore, PAR-1 and PAR-2 had opposing effects on the activation of ERK, p38, and NF-B.Conclusion. Activation of PAR-1 stimulates MMP-2 secretion, inhibits RASF growth and invasion, and decreases production of IL-17 and TNF␣ by RASFs, whereas activation of PAR-2 stimulates RASF growth and invasion and increases production of TNF␣. Thus, although PAR-1 and PAR-2 are coexpressed by RASFs, PAR-2 alone appears to be responsible for the aggressive properties of RASFs and is likely to contribute to the pathologic progression of RA.

Pompe disease is a lysosomal storage disorder caused by malfunctions of the acid alpha-glucosidase (GAA) enzyme with a consequent toxic accumulation of glycogen in cells. Muscle wasting and hypertrophic cardiomyopathy are the most common clinical signs that can lead to cardiac and respiratory failure within the first year of age in the more severe infantile forms. Currently available treatments have significant limitations and are not curative, highlighting a need for the development of alternative therapies. In this study, we investigated the use of a clinically relevant lentiviral vector to deliver systemically GAA through genetic modification of hematopoietic stem and progenitor cells (HSPCs). The overexpression of GAA in human HSPCs did not exert any toxic effect on this cell population, which conserved its stem cell capacity in xenograft experiments. In a murine model of Pompe disease treated at young age, we observed phenotypic correction of heart and muscle function with a significant reduction of glycogen accumulation in tissues after 6 months of treatment. These findings suggest that lentiviral-mediated HSPC gene therapy can be a safe alternative therapy for Pompe disease.