Nanosuspensions have emerged as a promising strategy for the efficient delivery of hydrophobic drugs because of their versatile features and unique advantages. Techniques such as media milling and highpressure homogenization have been used commercially for producing nanosuspensions. Recently, the engineering of nanosuspensions employing emulsions and microemulsions as templates has been addressed in the literature. The unique features of nanosuspensions have enabled their use in various dosage forms, including specialized delivery systems such as mucoadhesive hydrogels. Rapid strides have been made in the delivery of nanosuspensions by parenteral, peroral, ocular and pulmonary routes. Currently, efforts are being directed to extending their applications in site-specific drug delivery.

Previous studies demonstrated that targeted deletion of the Ron receptor tyrosine kinase (TK) domain in mice leads to marked hepatocyte protection in a well-characterized model of lipopolysaccharide (LPS)-induced acute liver failure in D-galactosamine (GalN)-sensitized mice. Hepatocyte protection in TK2/2 mice was observed despite paradoxically elevated serum levels of tumor necrosis factor alpha (TNF-a). To understand the role of Ron in the liver, purified populations of Kupffer cells and hepatocytes from wildtype (TK1/1) and TK2/2 mice were studied. Utilizing quantitative reverse-transcription polymerase chain reaction (RT-PCR), we demonstrated that Ron is expressed in these cell types. Moreover, we also recapitulated the protected hepatocyte phenotype and exaggerated cytokine production observed in the TK2/2 mice in vivo through the use of purified cultured cells ex vivo. We show that isolated TK2/2 Kupffer cells produce increased levels of TNF-a and select cytokines compared to TK1/1 cells following LPS stimulation. We also show that conditioned media from LPS-treated TK2/2 Kupffer cells was more toxic to hepatocytes than control media, suggesting the exaggerated levels of cytokines produced from the TK2/2 Kupffer cells are detrimental to wildtype hepatocytes. In addition, we observed that TK2/2 hepatocytes were more resistant to cell death compared to TK1/1 hepatocytes, suggesting that Ron functions in both the epithelial and inflammatory cell compartments to regulate acute liver injury. These findings were confirmed in vivo in mice with hepatocyte and macrophage cell-type-specific conditional Ron deletions. Mice with Ron loss selectively in hepatocytes exhibited less liver damage and increased survival compared to mice with Ron loss in macrophages. Conclusion: We dissected cell-type-specific roles for Ron such that this receptor modulates cytokine production from Kupffer cells and inhibits hepatocyte survival in response to injury. (HEPATOLOGY 2011;53:1618-1628

NFATc1 transcription factor is critical for lineage selection in T-cell differentiation, cardiac valve morphogenesis and osteoclastogenesis. We identified a role for calcineurin-NFAT signaling in lymphatic development and patterning. NFATc1 was colocalized with lymphatic markers Prox-1, VEGFR-3 and podoplanin on cardinal vein as lymphatic endothelial cells (LEC) are specified and as they segregate into lymph sacs and mature lymphatics. In NFATc1 null mice, Prox-1, VEGFR-3 and podoplanin positive endothelial cells sprouted from the cardinal vein at E11.5, but poorly coalesced into lymph sacs. NFAT activation requires the phosphatase calcineurin. Embryos treated in utero with the calcineurin inhibitor cyclosporine-A showed cytoplasmic NFATc1, diminished podoplanin and FGFR-3 expression by the lymphatics and irregular patterning of the LEC sprouts coming off the jugular lymph sac, which suggests a role for calcineurin-NFAT signaling in lymphatic patterning. In a murine model of injury-induced lymphangiogenesis, NFATc1 was expressed on the neolymphatics induced by lung-specific overexpression of VEGF-A. Mice lacking the calcineurin Aβ regulatory subunit, with diminished nuclear NFAT, failed to respond to VEGF-A with increased lymphangiogenesis. In vitro, endogenous and VEGF-A-induced VEGFR-3 and podoplanin expression by human microvascular endothelial cells was reduced by siRNA to NFATc1, to levels comparable to reductions seen with siRNA to Prox-1. In reporter assays, NFATc1 activated lymphatic specific gene promoters. These results demonstrate the role of calcineurin-NFAT pathway in lymphangiogenesis and suggest that NFATc1 is the principle NFAT involved.

Ron receptor kinase (MST1R) is important in promoting epithelial tumorigenesis, but the potential contributions of its specific expression in stromal cells have not been examined. Herein, we show that the Ron receptor is expressed in mouse and human stromal cells of the prostate tumor microenvironment. To test the significance of stromal Ron expression, prostate cancer cells were orthotopically implanted into the prostates of either wild type (WT) or Ron tyrosine kinase deficient (TK−/−; Mst1r−/−) hosts. In TK−/− hosts, prostate cancer cell growth was significantly reduced compared to tumor growth in TK+/+ hosts. Prostate tumors in TK−/− hosts exhibited an increase in tumor cell apoptosis, macrophage infiltration and altered cytokine expression. Reciprocal bone marrow transplantation studies and utilizing myeloid cell specific ablation of Ron demonstrated that loss of Ron in myeloid cells is sufficient to inhibit prostate cancer cell growth. Interestingly, depletion of CD8+ T-cells, but not CD4+ T-cells, was able to restore prostate tumor growth in hosts devoid of myeloid-specific Ron expression. These studies demonstrate a critical role for the Ron receptor in the tumor microenvironment, whereby Ron loss in tumor-associated macrophages inhibits prostate cancer cell growth, at least in part, by de-repressing the activity of CD8+ T cells.