Gene therapy targeted at the respiratory epithelium holds therapeutic potential for diseases such as cystic fibrosis and alpha-1 anti-trypsin deficiency. A variety of approaches such as intranasal or intratracheal instillation and aerosol delivery have been utilized to target genes to the airways. Polyethylenimine (PEI), a linear or branched polycationic polymer, has been used for delivery of genes to various organs. In this study, using fluorescein isothiocyanate (FITC)-labeled branched PEI, we initially examined the localization of PEI in the lungs after aerosol delivery to Balb/C mice. Further, after aerosol delivery of PEI-CAT DNA, in situ immunostaining for chloramphenicol acetyl transferase (CAT) protein was used to localize the transgene expression within the lungs. Immunohistochemistry for CAT, as well as localization of FITC-labeled PEI, revealed that after aerosol delivery, the PEI-DNA complexes deposit and subsequently transfect most of the epithelial cells in the conducting airways (including the peripheral airways). High levels of CAT were detected at 24 h after aerosol exposure and significant CAT expression was detected in the lungs up to 28 days after a single aerosol exposure. The data suggest that aerosol delivery of PEI-DNA complexes could be effective for the treatment of pulmonary diseases such as cystic fibrosis and alpha-1 anti-trypsin deficiency.

Hematopoietic stem cells (HSC) are unique in that they give rise both to new stem cells (self-renewal) and to all blood cell types. The cellular and molecular events responsible for the formation of HSC remain unknown mainly because no system exists to study it. Embryonic stem (ES) cells were induced to differentiate by coculture with the stromal cell line RP010 and the combination of interleukin (IL) 3, IL-6, and F (cell-free supernatants from cultures of the FLS4.1 fetal liver stromal cell line). Cell cytometry analysis of the mononuclear cells produced in the cultures was consistent with the presence of PgP-1+ Lin- early hematopoietic (B-220- Mac-1- JORO 75- TER 119-) cells and of fewer B-220+ IgM- B-cell progenitors and JORO 75+ T-lymphocyte progenitors. The cell-sorter-purified PgP-1+ Lin- cells produced by induced ES cells could repopulate the lymphoid, myeloid, and erythroid lineages of irradiated mice. The ES-derived PgP-1+ Lin- cells must possess extensive self-renewal potential, as they were able to produce hematopoietic repopulation of secondary mice recipients. Indeed, marrow cells from irradiated mice reconstituted (15-18 weeks before) with PgP-1+ Lin- cell-sorter-purified cells generated by induced ES cells repopulated the lymphoid, myeloid, and erythroid lineages of secondary mouse recipients assessed 16-20 weeks after their transfer into irradiated secondary mice. The results show that the culture conditions described here support differentiation of ES cells into hematopoietic cells with functional properties of HSC. It should now be possible to unravel the molecular events leading to the formation of HSC.

5% CO2 enrichment of air increased respiratory tract deposition of inhaled aerosol particles containing PTX and CPT.

The objective of this study was to assess the effect of cyclosporin A liposome aerosol on the anticancer activity of paclitaxel (PTX) liposome aerosol against renal cell carcinoma (Renca) pulmonary metastases in mice. Cyclosporin A (CsA) was administered as a liposome aerosol for one-half hour before starting one-half hour treatment with PTX liposome aerosol (CsA/PTX), and in a second groups of animals cyclosporin A liposome aerosol was given before PTX for one-half hour and also later by mixing a second dose of cyclosporin A aerosol with PTX aerosol and extending the treatment period to one hour (CsA/PTX + CsA). In one experiment, PTX and CsA/PTX aerosols were significantly more effective compared to untreated controls against renal cell cancer as measured by lung weights and tumor surface areas. CsA/PTX was significantly better that PTX alone as measured by lung weights and tumor area. In a second experiment, tumor areas of PTX and CsA/PTX treated mice were significantly reduced compared to untreated controls and CsA/PTX treated mice had significantly smaller tumor areas than PTX treated mice. In contrast, tumor numbers were not significantly fewer than controls in either therapeutic group. In a third experiment, tumor numbers and tumor areas were significantly fewer in mice treated with CsA/PTX and CsA/PTX + CsA compared to untreated controls. Mice treated with CsA/PTX + CsA had significantly fewer tumors and less tumor area than mice receiving CsA/PTX. While PTX treated mice were not different than untreated controls with respect to tumor numbers or tumor volumes, PTX treated mice had significantly greater tumor numbers and tumor areas than CsA/PTX and CsA/PTX + CsA treated mice. Co-administration of CsA with PTX demonstrated significant dose dependent anticancer effects against renal cell pulmonary metastases in mice. Toxicity manifested by weight loss was associated with the highest dose of CsA.

Martin E, Golunski E, Laing ST, Estrera AL, Sharina IG. Alternative splicing impairs soluble guanylyl cyclase function in aortic aneurysm. Am J Physiol Heart Circ Physiol 307: H1565-H1575, 2014. First published September 19, 2014 doi:10.1152/ajpheart.00222.2014 receptor soluble guanylyl cyclase (sGC) is a key regulator of several important vascular functions and is important for maintaining cardiovascular homeostasis and vascular plasticity. Diminished sGC expression and function contributes to pathogenesis of several cardiovascular diseases. However, the processes that control sGC expression in vascular tissue remain poorly understood. Previous work in animal and cell models revealed the complexity of alternative splicing of sGC genes and demonstrated its importance in modulation of sGC function. The aim of this study was to examine the role of alternative splicing of ␣1 and ␤1 sGC in healthy and diseased human vascular tissue. Our study found a variety of ␣1 and ␤1 sGC splice forms expressed in human aorta. Their composition and abundance were different between samples of aortic tissue removed during surgical repair of aortic aneurysm and samples of aortas without aneurysm. Aortas with aneurysm demonstrated decreased sGC activity, which correlated with increased expression of dysfunctional sGC splice variants. In addition, the expression of 55-kDa oxidation-resistant ␣1 isoform B sGC (␣1-IsoB) was significantly lower in aortic samples with aneurysm. The ␣1-IsoB splice variant was demonstrated to support sGC activity in aortic lysates. Together, our results suggest that alternative splicing contributes to diminished sGC function in vascular dysfunction. Precise understanding of sGC splicing regulation could help to design new therapeutic interventions and to personalize sGC-targeting therapies in treatments of vascular disease. nitric oxide; soluble guanylyl cyclase; alternative splicing; aorta