Rescue Effects and Underlying Mechanisms of Intragland Shh Gene Delivery on Irradiation-Induced Hyposalivation

Abstract

Irreversible hypofunction of salivary glands is common in head and neck cancer survivors treated with radiotherapy and can only be temporarily relieved with current treatments. We found in an inducible sonic hedgehog (Shh) transgenic mouse model that transient activation of the Hedgehog pathway after irradiation rescued salivary gland function in males by preserving salivary stem/progenitor cells and parasympathetic innervation. To translate these findings into feasible clinical application, we evaluated the effects of Shh gene transfer to salivary glands of wild-type mice on irradiation-induced hyposalivation. Shh or control GFP gene was delivered by noninvasive retrograde ductal instillation of corresponding adenoviral vectors. In both male and female mice, Shh gene delivery efficiently activated Hedgehog/Gli signaling, and significantly improved stimulated saliva secretion and preserved saliva-producing acinar cells after irradiation. In addition to preserving parasympathetic innervation through induction of neurotrophic factors, Shh gene delivery also alleviated the irradiation damage of the microvasculature, likely via inducing angiogenic factors, but did not expand the progeny of cells responsive to Hedgehog/Gli signaling. These data indicate that transient activation of the Hedgehog pathway by gene delivery is promising to rescue salivary function after irradiation in both sexes, and the Hedgehog/Gli pathway may function mainly in cell nonautonomous manners to achieve the rescue effect.

Figure 1.

Transient activation of the Hh/Gli pathway in SMGs by retrograde ductal instillation of Ad-Shh. Ad-GFP or Ad-Shh was delivered into SMGs of male and female Ptch1-lacZ mice by retrograde ductal instillation. SMGs were collected 3 or 10 days after Ad delivery and subjected to X-Gal staining of sections (A) or qRT-PCR analysis of the expression of rat Shh (rShh) transgene or Hh target gene Gli1 (B). **p < 0.01 versus the nontreated (NT) group.

Figure 2.

Delivery of Shh gene into SMGs partially rescued irradiation-induced hyposalivation. Male and female C57BL/6 mice were subjected to 15 Gy of irradiation in the neck region and retrograde ductal instillation of Ad-GFP or Ad-Shh 3 or 30 days later. The stimulated whole saliva flow rate was measured on day 90 after IR and normalized with body weight and then with saliva flow rate of age-matched NT mice (A). SMGs were collected 90 days after IR and analyzed by qRT-PCR (B) or Western blotting (C) for the expression of acinar marker Aqp5. *p < 0.05. IR, irradiation.

Figure 3.

Transient Hh activation alleviated IR damage to microvascular endothelial cells by upregulating angiogenic factors. (A) Western blot of Aqp1 in SMGs 90 days after IR and quantitative analysis of relative Aqp1 protein level normalized with GAPDH protein level (means ± SEM, n = 3). (B) Representative immunofluorescence staining of the endothelial marker Aqp1 in male SMGs and quantification of Aqp1⁺ microvessel density in SMGs 90 days after IR (95% confidence intervals shown in a box plot; the upper and lower boundaries of the box represent the 75th and 25th percentiles of the number of Aqp1⁺ microvessels per field per mouse, and the horizontal line within the box represents the median value). (C) qRT-PCR analysis of the expression of genes related to angiogenesis in SMGs collected 7 days after Ad instillation or 10 days after IR (n = 3). *p < 0.05. Ad, adenovirus.

Figure 4.

Intragland Shh gene delivery rescued parasympathetic innervation of SMGs. (A) SMGs collected 90 days after IR were analyzed with Western blot for expression of Gfra2, a makers of parasympathetic innervation. (B) Representative staining of acetylcholinesterase (AChE) activities on male SMG sections 90 days after IR and the quantification of relative AChE⁺ areas normalized to NT. (C) SMGs collected 10 days after IR or 7 days after Ad instillation were analyzed with qRT-PCR assay for the mRNA expression of neurotrophic factors (n = 3). *p < 0.05.

Figure 5.

Distribution of progeny of Bmi1⁺ cells during functional regeneration or after IR with or without transient Hh activation. SMGs were collected 21 days after duct ligation (A) or 90 days after IR (B) from Bmi1-CreER/Rosa26R-lacZ mice treated with 4-OHT after duct ligation or Ad-Shh distillation. Frozen sections of these SMG samples were analyzed by X-Gal staining for activity of the lacZ/β-Gal reporter or by double-immunofluorescence staining for the expression of Aqp5 and β-Gal reporter.