The treatment of most head and neck cancer patients includes ionizing radiation (IR). Salivary glands in the IR field suffer irreversible damage. Previously, we reported that adenoviral (Ad)-mediated transfer of the human aquaporin-1 (hAQP1) cDNA to rat submandibular glands following IR restored salivary flow to near normal levels. It is unclear if this strategy is useful in larger animals. Herein, we evaluated AdhAQP1-mediated gene transfer after parotid gland IR (20 Gy) in the miniature pig. Sixteen weeks following IR, salivation from the targeted gland was decreased by >80%. AdhAQP1 administration resulted in a dose-dependent increase in parotid salivary flow to approximately 80% of pre-IR levels on day 3. A control Ad vector was without significant effect. The effective AdhAQP1 dose was 2.5 x 10(5) pfu/microl infusate, a dose that leads to comparable transgene expression in murine and minipig salivary glands. Three days after Ad vector administration little change was observed in clinical chemistry and hematology values. These findings demonstrate that localized delivery of AdhAQP1 to IR-damaged salivary glands increases salivary secretion, without significant general adverse events, in a large animal model.

Exploring the molecular mechanisms underlying directed differentiation is helpful in the development of clinical applications of mesenchymal stem cells (MSCs). Our previous study on dental tissue-derived MSCs demonstrated that secreted frizzled-related protein 2 (SFRP2), a Wnt inhibitor, could enhance osteogenic differentiation in stem cells from the apical papilla (SCAPs). However, how SFRP2 promotes osteogenic differentiation of dental tissue-derived MSCs remains unclear. In this study, we used SCAPs to investigate the underlying mechanisms. SCAPs were isolated from the apical papilla of immature third molars. Western blot and real-time RT-PCR were applied to detect the expression of β-catenin and Wnt target genes. Alizarin Red staining, quantitative calcium analysis, transwell cultures and in vivo transplantation experiments were used to study the osteogenic differentiation potential of SCAPs. SFRP2 inhibited canonical Wnt signaling by enhancing phosphorylation and decreasing the expression of nuclear β-catenin in vitro and in vivo. In addition, the target genes of the Wnt signaling pathway, AXIN2 (axin-related protein 2) and MMP7 (matrix metalloproteinase-7), were downregulated by SFRP2. WNT1 inhibited the osteogenic differentiation potential of SCAPs. SFRP2 could rescue this WNT1-impaired osteogenic differentiation potential. The results suggest that SFRP2 could bind to locally present Wnt ligands and alter the balance of intracellular Wnt signaling to antagonize the canonical Wnt pathway in SCAPs. This elucidates the molecular mechanism underlying the SFRP2-mediated directed differentiation of SCAPs and indicates potential target genes for improving dental tissue regeneration.

Patients frequently experience a loss of salivary function following irradiation (IR) for the treatment of an oral cavity and oropharyngeal cancer. Herein, we tested if transfer of fibroblast growth factor-2 (FGF2) cDNA could limit salivary dysfunction after fractionated IR (7.5 or 9 Gy for 5 consecutive days to one parotid gland) in the miniature pig (minipig). Parotid salivary flow rates steadily decreased by 16 weeks post-IR, whereas blood flow in the targeted parotid gland began to decrease ~3 days after beginning IR. By 2 weeks, post-IR salivary blood flow was reduced by 50%, at which point it remained stable for the remainder of the study. The single preadministration of a hybrid serotype 5 adenoviral vector encoding FGF2 (AdLTR₂EF1a-FGF2) resulted in the protection of parotid microvascular endothelial cells from IR damage and significantly limited the decline of parotid salivary flow. Our results suggest that a local treatment directed at protecting salivary gland endothelial cells may be beneficial for patients undergoing IR for oral cavity and oropharyngeal cancer.