Elsevier

Journal of Controlled Release

Volume 233, 10 July 2016, Pages 191-197
Journal of Controlled Release

Pulmonary administration of 1,25-dihydroxyvitamin D3 to the lungs induces alveolar regeneration in a mouse model of chronic obstructive pulmonary disease

https://doi.org/10.1016/j.jconrel.2016.05.006Get rights and content

Abstract

Chronic obstructive pulmonary disease (COPD) is a progressive respiratory disease with several causes, including smoking, and no curative therapeutic agent is available, particularly for destructive alveolar lesions. In this study, we investigated the differentiation-inducing effect on undifferentiated lung cells (Calu-6) and the alveolar regenerative effect of the active vitamin 1,25-dihydroxy vitamin D3 (VD3) with the ultimate goal of developing a novel curative drug for COPD. First, the differentiation-inducing effect of VD3 on Calu-6 cells was evaluated. Treatment with VD3 increased the proportions of type I alveolar epithelial (AT-I) and type II alveolar epithelial (AT-II) cells constituting alveoli in a concentration- and treatment time-dependent manner, demonstrating the potent differentiation-inducing activity of VD3 on Calu-6 cells. We thus administered VD3 topically to the mice lung using a previously developed intrapulmonary administration via self-inhalation method. To evaluate the alveolus-repairing effect of VD3, we administered VD3 intrapulmonarily to elastase-induced COPD model mice and computed the mean distance between the alveolar walls as an index of the extent of alveolar injury. Results showed significant decreases in the alveolar wall distance in groups of mice that received 0.01, 0.1, and 1 μg/kg of intrapulmonary VD3, revealing excellent alveolus-regenerating effect of VD3. Furthermore, we evaluated the effect of VD3 on improving respiratory function using a respiratory function analyzer. Lung elasticity and respiratory competence [forced expiratory volume (FEV) 1 s %] are reduced in COPD, reflecting advanced emphysematous changes. In elastase-induced COPD model mice, although lung elasticity and respiratory competence were reduced, VD3 administered intrapulmonarily twice weekly for 2 weeks recovered tissue elastance and forced expiratory volume in 0.05 s to the forced vital capacity, which are indicators of lung elasticity and respiratory competence, respectively, to levels comparable to those in normal mice. These results revealed the potent activity of VD3 in inducing differentiation of the Calu-6 cells and the effect of topical administration of VD3 to the lungs to induce lung regeneration at histological and functional levels, demonstrating the potential of VD3 as a curative agent for alveolar destruction in COPD.

Introduction

Chronic obstructive pulmonary disease (COPD) is a disease that collectively refers to conventional chronic bronchitis and emphysema [1]. According to an epidemiological survey, COPD is the fourth leading cause of death worldwide, and the estimated number of patients has been reported to be 329 million [2]. In COPD, inflammation occurs in the lungs due to long-term inhalation of toxic gas, primarily tobacco smoke, causing coughing, sputum production, and bronchitis, and eventually, emphysema, in which alveoli are destroyed [3]. Among COPD pathologies, emphysematous changes that show irreversible destruction of alveoli are the primary cause of the reduced respiratory function [3]. However, no therapeutic drug is currently available to completely cure these alveolar-destructive pathological changes.

In the recent years, focus on regenerative therapy for the purpose of repairing damaged tissues has been growing. The alveolus, the target of regenerative therapy in this study, comprises the alveolar space, in which gas is retained, and the alveolar epithelium surrounding it. The alveolar epithelium comprises type I alveolar epithelial (AT-I) cells and type II alveolar epithelial (AT-II) cells. AT-I cells form the blood–air barrier with capillary endothelial cells and basement membranes surrounding the alveoli and exchange intra-alveolar gas and blood gas. AT-II cells contain an abundance of lamellated corpuscles, which exocytotically release the pulmonary surfactant and form the alveolar coating layer. We were the first to report that the extracellular matrix-associated factor integrin [4] and the synthetic retinoid Am80 [5] and Wortmannin [6] are potent regenerate alveoli. The discovery of a molecule responsible for the regeneration of alveoli has opened the path toward alveolar regenerative therapy that had previously been impossible, and identification of more alveolus-regenerating molecules is anticipated.

In this study, we focused on vitamin D, a lipophilic vitamin linked to inflammatory pathologies of COPD. Vitamin D is further divided into vitamin D2 (ergocalciferol) and vitamin D3 (cholecalciferol). Vitamin D3 is found abundantly in animals and plays important roles in physiological events in humans, such as osteogenesis [7]. Vitamin D3, as the active form of vitamin D (calcitriol or 1,25-dihydroxycholecalciferol), increases blood calcium levels and regulates proliferation and differentiation of cells [8]. Vitamin D binds to vitamin D-binding protein (DBP) in blood and acts on the intranuclear receptor vitamin D receptor (VDR) to control the transcription of target genes. Epidemiological survey data have shown that tobacco smoke — the major causative factor of COPD — decreases vitamin D levels, and tobacco extracts have been reported to suppress gene transcription induced by the active form of vitamin D3 via the nuclear receptor VDR in alveolar epithelial cells [9], [10]. Blood vitamin D levels and DBP expression levels have been reported to correlate with decreased respiratory function in COPD, and inflammation has been shown to occur in the lungs of VDR-knockout mice [11], [12], [13], [14]. However, whether active-form vitamin D3 is related to differentiation of the Calu-6 cells or regeneration of alveoli has not been reported.

The half-life of active-form vitamin D3 in the blood is approximately 15 h, posing a major problem because the pharmacological effect in the lungs cannot be assessed completely with systemic administration. Intrapulmonary administration is a very effective drug delivery method for evaluating drugs that exert their pharmacological effects in the lungs [6]. Conventional devices for intrapulmonary administration have been used for forced administration; however, they can damage the lungs and are technically demanding. We have previously established a novel method of intrapulmonary administration via self-inhalation by mice, and the method can deliver drugs efficiently and topically to the lungs [4], [5], [6].

In this study, we aimed to evaluate the ability of the active form of vitamin D3 1,25-dihydroxyvitamin D3 (VD3) to induce differentiation of the Calu-6 cells and the alveolus-regenerating effect of intrapulmonarily administered VD3.

Section snippets

Animals

Male ICR mice were purchased from Sankyo Labo Service Corporation (Tokyo, Japan). Animals were housed in a temperature-controlled (24 ± 1 °C) facility maintained on a light:dark 12-h cycle with standard food available ad libitum. All animal procedures followed the guidelines established by the Animal Care and Use Committee of the Tokyo University of Science (No. Y12058, Y13008, Y14052, Y15042).

Cell culture

The Calu-6 cells (ATCC® HTB-56™) were cultured EMEM (Sigma-Aldrich, St. Louis, MO) supplemented with 10%

Verification of the differentiation-inducing effect of VD3 on the Calu-6 cells

To assess the differentiation-inducing effect of VD3 on the Calu-6 cells, we counted cells demonstrating positive immunostaining for CD90, aquaporin-5 (AQP-5), and surfactant protein-A (SP-A), which are specific markers of the Calu-6 cells, AT-I cells, and AT-II cells, respectively. Results showed that VD3 induced differentiation into AT-I and AT-II cells in a concentration- and treatment time-dependent manner (Fig. 1). In a 6-day culture with 10 μM of VD3 present, cell subpopulations that

Discussion

The most significant finding in this study was demonstration of the potential of VD3 as a novel curative therapeutic agent for destructive alveolar lesions in COPD. The involvement of VD3 in regeneration of alveoli has not previously been reported. Using the recently established Calu-6 cells, we revealed in this study that VD3 was a potent differentiation inducer for Calu-6 cells. Moreover, that topical administration of VD3 to the lungs induced the regeneration of alveoli and the recovery of

Conclusion

In this study, VD3 topically administered to the lungs induce lung regeneration at histological and functional levels. The results from this study suggest that VD3 holds the potential to serve as a curative agent for destructive alveolar changes in COPD.

Acknowledgments

This study was partially supported by the Grant-in-Aid for Research Activity Start-up [Michiko Horiguchi, 24890257], and the Grant-in-Aid for Young Scientists (B) [Michiko Horiguchi, 25860029] from the Japan Society for the Promotion of Science.

References (15)

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