93. Increased Number of Lung CD34+ Sca-1+ Putative Stem Cells Mediated by Adenovirus Overexpression by Sonic Hedgehog in the Respiratory Tract of Adult Mice

Abstract

Sonic hedgehog (Shh), a mammalian hedgehog pathway ligand that plays an important role in the development of mammalian epithelia, is a mediator of embryonic lung development, including regeneration of respiratory epithelium. Shh is synthesized as a 47kDa precursor, which under goes autocatalytic cleavage and loss of the carboxy (C)-terminal to generate the 19kDa amino (N)-terminal Shh that is secreted and is responsible for all known biological activities of the molecule. Based on the knowledge that the hedgehog pathways are implicated in the maintenance of stem or progenitor cells of many adult tissues, we hypothesized that overexpression of Shh in the lung would be associated with increased numbers of stem cells in the respiratory tract. To assess this concept, we administered to the lung an adenovirus (AdShhN) encoding a genetically engineered form of the N-terminal of Shh (ShhN) that lacks the enzymatic activity in the C-terminal for the addition of cholesterol, rendering ShhN more diffusible. AdShhN was administered to the respiratory tract of adult C57BL/6 mice via the intratracheal route, with naive mice or mice that received AdNull control vector with no transgene, serving as controls. Based on current knowledge of lung stem cells that are multipotent and have self-renewing properties, lung suspensions were assessed over time for cells that are negative for CD45 and pecam (CD45neg pecamneg; thus excluding the hematopoietic and endothelial lineages), and positive for the stem cell markers CD34 and Sca-1 (CD34pos, Sca-1pos). The number of CD34pos, Sca-1pos cells in the CD45neg pecamneg population were quantified in lung suspensions 3, 7 and 10 days following administration of the Ad vectors by flow cytometry. In naive mice, the Sca-1pos, CD45neg pecamneg cells represented 0.5 -1 % of the total lung cell preparation. After 3 days, in the mice who had received AdShhN, 25 |[plusmn]| 2 % of the Sca-1pos, CD45neg pecamneg cells were positive for CD34 compared to 9 |[plusmn]| 3 % for the naive and 11 |[plusmn]| 4 % for the AdNull groups (p<0.05, both comparisons). At 7 days, the number of CD34pos, Sca-1pos, CD45neg pecamneg cells in the AdhhN group was increased 4- and 3-fold compared to the naive and AdNull group, respectively (p<0.01, both comparisons). At 10 days, although still significantly higher than controls the CD34pos, Sca-1pos, CD45neg cells had returned towards the resting values 1.6- and 1.7-fold in the AdShhN group compared to naive or AdNull, respectively (p<0.01). The data demonstrates that overexpression of ShhN in the respiratory tract increases the number of pulmonary CD34pos, Sca-1pos, CD45neg pecamneg cells, an observation that has implications relevant to the biology of repair of the lung, as well as the development of strategies enhancing lung repair.