BRCA1 and 53BP1 regulate reprogramming efficiency by mediating DNA repair pathway choice at replication-associated double-strand breaks.

Reprogramming to pluripotency is associated with DNA damage and requires the functions of the BRCA1 tumor suppressor. Here, we leverage separation-of-function mutations in BRCA1/2 as well as the physical and/or genetic interactions between BRCA1 and its associated repair proteins to ascertain the relevance of homology-directed repair (HDR), stalled fork protection (SFP), and replication gap suppression (RGS) in somatic cell reprogramming. Surprisingly, loss of SFP and RGS is inconsequential for the transition to pluripotency. In contrast, cells deficient in HDR, but proficient in SFP and RGS, reprogram with reduced efficiency. Conversely, the restoration of HDR function through inactivation of 53bp1 rescues reprogramming in Brca1-deficient cells, and 53bp1 loss leads to elevated HDR and enhanced reprogramming in mouse and human cells. These results demonstrate that somatic cell reprogramming is especially dependent on repair of replication-associated double-strand breaks (DSBs) by the HDR activity of BRCA1 and BRCA2 and can be improved in the absence of 53BP1.

Inhibition of Tumor-Derived C-C Motif Chemokine Ligand 2 Expression Attenuates Tactile Allodynia in NCTC 2472 Fibrosarcoma-Inoculated Mice.

Neuropathic pain associated with cancers is caused by tumor growth compressing and damaging nerves, which would also be enhanced by inflammatory factors through sensitizing nociceptor neurons. A troublesome hallmark symptom of neuropathic pain is hypersensitivity to innocuous stimuli, a condition known as "tactile allodynia", which is often refractory to NSAIDs and opioids. The involvement of chemokine CCL2 (monocyte chemoattractant protein-1) in cancer-evoked neuropathic pain is well established, but opinions remain divided as to whether CCL2 is involved in the production of tactile allodynia with tumor growth. In this study, we constructed Ccl2 knockout NCTC 2472 (Ccl2-KO NCTC) fibrosarcoma cells and conducted pain behavioral test using Ccl2-KO NCTC-implanted mice. Implantation of naïve NCTC cells around the sciatic nerves of mice produced tactile allodynia in the inoculated paw. Although the growth of Ccl2 KO NCTC-formed tumors was comparable to that of naïve NCTC-formed tumors, Ccl2-KO NCTC-bearing mice failed to show tactile pain hypersensitivity, suggesting the involvement of CCL2 in cancer-induced allodynia. Subcutaneous administration of controlled-release nanoparticles containing the CCL2 expression inhibitor NS-3-008 (1-benzyl-3-hexylguanidine) significantly attenuated tactile allodynia in naïve NCTC-bearing mice accompanied by a reduction of CCL2 content in tumor masses. Our present findings suggest that inhibition of CCL2 expression in cancer cells is a useful strategy to attenuate tactile allodynia induced by tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for the treatment of cancer-evoked neuropathic pain. SIGNIFICANCE STATEMENT: The blockade of chemokine/receptor signaling, particularly for C-C motif chemokine ligand 2 (CCL2) and its high-affinity receptor C-C chemokine receptor type 2 (CCR2), has been implicated to attenuate cancer-induced inflammatory and nociceptive pain. This study demonstrated that continuous inhibition of CCL2 production from cancer cells also prevents the development of tactile allodynia associated with tumor growth. Development of a controlled-release system of CCL2 expression inhibitor may be a preventative option for management of cancer-evoked tactile allodynia.

Decreased ZO1 expression causes loss of time-dependent tight junction function in the liver of ob/ob mice.

Diabetes patients are at a high risk of developing complications related to angiopathy and disruption of the signal transduction system. The liver is one of the multiple organs damaged during diabetes. Few studies have evaluated the morphological effects of adhesion factors in diabetic liver. The influence of diurnal variation has been observed in the expression and functioning of adhesion molecules to maintain tissue homeostasis associated with nutrient uptake. The present study demonstrated that the rhythm-influenced functioning of tight junction was impaired in the liver of ob/ob mice. The tight junctions of hepatocytes were loosened during the dark period in control mice compared to those in ob/ob mice, where the hepatocyte gaps remained open throughout the day. The time-dependent expression of zonula occludens 1 (ZO1, encoded by Tjp1 gene) in the liver plays a vital role in the functioning of the tight junction. The time-dependent expression of ZO1 was nullified and its expression was attenuated in the liver of ob/ob mice. ZO1 expression was inhibited at the mRNA and protein levels. The expression rhythm of ZO1 was found to be regulated by heat shock factor (HSF)1/2, the expression of which was reduced in the liver of ob/ob mice. The DNA-binding ability of HSF1/2 was decreased in the liver of ob/ob mice compared to that in control mice. These findings suggest the involvement of impaired expression and functioning of adhesion factors in diabetic liver complications.

AMFR and DCTN2 genes cause transplantation resistance of adipose-derived mesenchymal stem cells in type 1 diabetes mellitus.

Type 1 diabetes mellitus (T1DM) is characterized by pancreatic beta cell destruction by autoantibodies and other factors, resulting in insulin secretion deficiency. Therefore, beta cell regeneration would be necessary to cure the disease. Nevertheless, the impact of type 1 diabetes on the stemness and transplantation efficiency of stem cells has not been previously described. In this study, we used next-generation sequencing to identify genes differentially expressed in T1DM adipose-derived stem cells (T1DM ADSCs) that originate from patients with type 1 diabetes. Furthermore, we evaluated their effects on transplantation efficiency following xenotransplantation into immunodeficient mice. In the T1DM ADSCs transplant group, the volume and weight of the graft were significantly reduced and the transplant efficiency was reduced. Next-generation sequencing and quantitative PCR results showed that T1DM ADSCs had significantly increased expression of AMFR and DCTN2. AMFR and DCTN2 gene knockdown in T1DM ADSC significantly restored cell proliferation and stem cell marker expression. Therefore, transplantation of T1DM ADSCs, in which AMFR and DCTN2 were knocked down, into immunodeficient mice improved transplant efficiency. This study revealed that AMFR and DCTN2 can reduce transplantation efficiency of T1DM ADSCs. Focusing on AMFR and DCTN2 is expected to increase the efficiency of stem cell transplantation therapy for diabetic patients.

Potential mechanism of hepatic lipid accumulation during a long-term rest phase restricted feeding in mice.

Eating during a rest phase disrupts the biological clock system and leads to obesity and metabolic diseases. Although a rest phase restricted feeding (RF) is reported to enhance hepatic lipid accumulation, the mechanism(s) of the phenomenon is still unknown. This study evaluated the potential involvement of the CD36-related transport of lipids into the liver in mice with the RF procedure. This study showed that hepatic lipid accumulation was more significant in the RF group compared with mice under an active phase restricted feeding (AF). The RF procedure also elevated the expression of CD36 mRNA and its protein on the cellular membrane throughout the day. The transcription factor profiling array revealed that the RF activated the proliferator-activated receptor-γ (PPARγ), one of the CD36 transcript enhancers. In the liver of RF mice, the expression of miR-27b-3p, which is known to interfere with PPARγ gene expression, significantly decreased. These results suggest that the RF procedure inhibits the expression of miR-27b-3p in the liver and subsequently elevates PPARγ activity. Activated PPARγ might lead to CD36 upregulation, which, in turn, stimulates the transport of lipids into the liver.

A Pragmatic Pilot Cluster-Randomized Study of Tobacco Screening and Smoking Cessation Program for Community Pharmacies in Japan: FINE Program.

OBJECTIVES: To investigate the effectiveness of a smoking cessation program (FINE program) in community pharmacies. METHODS: A cluster-randomized controlled trial was conducted in 11 community pharmacies in Japan. The participants were randomly assigned to a pharmacist-led structured smoking cessation program (intervention group) or pharmacist-led usual care (control group). The intervention group was followed up over the telephone on the third day of smoking cessation, and ongoing follow-up and advice were provided according to the original smoking cessation guidebook developed for the current study based on a behavioral change approach. The control group received brief advice and ready-made pamphlets on smoking cessation from pharmacists upon their visit to these community pharmacies. The primary outcome was continued smoking cessation as determined by self-reporting and carbon monoxide monitoring with a microsmokerlyzer after 3 months. RESULTS: Five hundred and seventy-two smokers who met the eligibility criteria visited the pharmacies included in the study. Of these individuals, 24 patients agreed to participate in the study. The quit rates were 45.5% and 18.2% in the intervention and control groups, respectively (P = 0.380, effect size = 0.60). CONCLUSION: Based on the effect size values, the FINE program may be effective to some extent, but the difference was not significant. We speculate that this is related to the small sample size due to difficulty in recruiting. Further studies with an effective recruitment method and larger sample sizes are needed to accurately verify the effectiveness of this program.

Exosome Degeneration in Mesenchymal Stem Cells Derived from Patients with Type 1 Diabetes Mellitus.

Type 1 diabetes mellitus is characterized by the destruction of pancreatic ß-cells and requires the regeneration of these destroyed pancreatic ß-cells for radical treatment. The degeneration of organelles in stem cells compromises stem cell quality; however, organelles in the mesenchymal stem cells of patients with type 1 diabetes mellitus have not been characterized previously. In this study, we use transmission electron microscopy to evaluate the degeneration of organelles in adipose-derived stem cells of patients with type 1 diabetes mellitus (T1DM ADSCs). Compared to adipose-derived stem cells from healthy humans, T1DM ADSCs degenerate differently, characterized by prominent enlarged spherical vesicles. The exosomes of T1DM ADSCs are found to be enlarged, reduced in number, and increased in the percentage of those positive for tetraspanin CD9. The findings of this study provide insight into the characteristics of stem cells in patients with type 1 diabetes mellitus.

The Transplantation Resistance of Type II Diabetes Mellitus Adipose-Derived Stem Cells Is Due to G6PC and IGF1 Genes Related to the FoxO Signaling Pathway.

In cases of patients with rapidly progressive diabetes mellitus (DM), autologous stem cell transplantation is considered as one of the regenerative treatments. However, whether the effects of autonomous stem cell transplantation on DM patients are equivalent to transplantation of stem cells derived from healthy persons is unclear. This study revealed that adipose-derived mesenchymal stem cells (ADSC) derived from type II DM patients had lower transplantation efficiency, proliferation potency, and stemness than those derived from healthy persons, leading to a tendency to induce apoptotic cell death. To address this issue, we conducted a cyclopedic mRNA analysis using a next-generation sequencer and identified G6PC3 and IGF1, genes related to the FoxO signaling pathway, as the genes responsible for lower performance. Moreover, it was demonstrated that the lower transplantation efficiency of ADSCs derived from type II DM patients might be improved by knocking down both G6PC3 and IGF1 genes. This study clarified the difference in transplantation efficiency between ADSCs derived from type II DM patients and those derived from healthy persons and the genes responsible for the lower performance of the former. These results can provide a new strategy for stabilizing the quality of stem cells and improving the therapeutic effects of regenerative treatments on autonomous stem cell transplantation in patients with DM.

Characterizing the degeneration of nuclear membrane and mitochondria of adipose-derived mesenchymal stem cells from patients with type II diabetes.

Regenerative therapeutic approaches involving the transplantation of stem cells differentiated into insulin-producing cells are being studied in patients with rapidly progressing severe diabetes. Adipose-derived mesenchymal stem cells have been reported to have varied cellular characteristics depending on the biological environment of the location from which they were harvested. However, the characteristics of mesenchymal stem cells in type II diabetes have not been clarified. In this study, we observed the organelles of mesenchymal stem cells from patients with type II diabetes under a transmission electron microscope to determine the structure of stem cells in type II diabetes. Transmission electron microscopic observation of mesenchymal stem cells from healthy volunteers (N-ADSC) and those from patients with type II diabetes (T2DM-ADSC) revealed enlarged nuclei and degenerated mitochondrial cristae in T2DM-ADSCs. Moreover, T2DM-ADSCs were shown to exhibit a lower expression of Emerin, a constituent protein of the nuclear membrane, and a decreased level of mitochondrial enzyme activity. In this study, we successfully demonstrated the altered structure of nuclear membrane and the decreased mitochondrial enzyme activity in adipose-derived mesenchymal cells from patients with type II diabetes. These findings have contributed to the understanding of type II diabetes-associated changes in mesenchymal stem cells used for regenerative therapy.

Pharmacist-Delivered Smoking Cessation Program in Community Pharmacy (The FINE Program) in Japan-The Development of a Training Course and a Feasibility Study.

AIM: Although smoking cessation support has been recommended as a routine component of pharmacists'duties, there is limited evidence of smoking cessation being achieved successfully in community pharmacy. The aim of this study was to develop a new smoking cessation program for use in the community pharmacy setting and investigate its feasibility. METHODS: A feasibility study (the Family pharmacist's Intervention for Nicotine Elimination [FINE] program) was conducted using 8 pharmacists at 2 community pharmacies in Japan. The pharmacists recruited as subjects smokers 20 or more years of age who were taking medications such as antidiabetes drugs. The patients completed questionnaires assessing their smoking status, and the pharmacists provided them with smoking cessation support services. Participating patients met with the pharmacists or talked to them on the phone 5 times at 2- to 4-week intervals and received personalized and structured brief smoking cessation advice. The primary outcome was continuous abstinence determined by Micro Smokerlyzer carbon monoxide monitor at 3 months. RESULTS: Of 5306 patients, 2296 patients were screened and the rate of smoking was found to be 12.7%. Five smoking patients received the FINE program from pharmacists who had received training. One of the 5 succeeded in quitting smoking after 3 months. CONCLUSIONS: This is the first study to target Japanese smoking patients in community pharmacies with a brief structured intervention. The results tentatively support the feasibility of the FINE program. Further research including a randomized controlled trial is required to confirm the effectiveness of the FINE program.

Induction of Dbp by a histone deacetylase inhibitor is involved in amelioration of insulin sensitivity via adipocyte differentiation in ob/ob mice.

We previously reported that a histone deacetylase inhibitor (HDACi) increases D-site binding protein (Dbp) mRNA expression in adipose tissue and subsequently improved insulin sensitivity of obese (ob/ob) mice. However, the potential mechanism of this phenomenon was unclear. Thus, the aim of this study was to clarify the molecular mechanism involved in enhanced Dbp mRNA expression and improvement of insulin sensitivity in mice. Ob/ob mice were treated with HDACi every second day for 3 weeks. At the end of treatment, an insulin tolerance test was performed and epididymal adipose tissue obtained for fractionation into adipocytes and preadipocytes. HDACi improved insulin sensitivity in ob/ob mice and significantly increased Dbp mRNA in epididymal adipose tissue. Further, epididymal adipocytes of ob/ob mice showed a tendency towards a larger size distribution, while HDACi increased the proportion of smaller sized cells in fractionated preadipocytes. Dbp knocked-down 3T3-L1 cells down-regulated peroxisome proliferator-activated receptor-γ (PPAR-γ1) protein expression during adipogenesis, which suppressed adipocyte differentiation. These data indicate that DBP promotes adipocyte differentiation via direct up-regulation of PPAR-γ1 production in preadipocytes. In fractionated preadipocytes of ob/ob mice, DBP binding to the promoter region of the Ppar-γ gene and splicing variant of Ppar-γ (Ppar-γ1sv) mRNA expression were suppressed. HDACi significantly increased DBP binding to the Ppar-γ gene and Ppar-γ1sv transcription. Altogether, this indicates a modification in genetic regulation downstream from the circadian clock that can ameliorate an environmental function of adipose tissue, leading to improved insulin sensitivity in ob/ob mice.

The BRCT Domains of the BRCA1 and BARD1 Tumor Suppressors Differentially Regulate Homology-Directed Repair and Stalled Fork Protection.

The BRCA1 tumor suppressor preserves genome integrity through both homology-directed repair (HDR) and stalled fork protection (SFP). In vivo, BRCA1 exists as a heterodimer with the BARD1 tumor suppressor, and both proteins harbor a phosphate-binding BRCT domain. Here, we compare mice with mutations that ablate BRCT phospho-recognition by Bard1 (Bard1S563F and Bard1K607A) or Brca1 (Brca1S1598F). Brca1S1598F abrogates both HDR and SFP, suggesting that both pathways are likely impaired in most BRCA1 mutant tumors. Although not affecting HDR, the Bard1 mutations ablate poly(ADP-ribose)-dependent recruitment of BRCA1/BARD1 to stalled replication forks, resulting in fork degradation and chromosome instability. Nonetheless, Bard1S563F/S563F and Bard1K607A/K607A mice, unlike Brca1S1598F/S1598F mice, are not tumor prone, indicating that HDR alone is sufficient to suppress tumor formation in the absence of SFP. Nevertheless, because SFP, unlike HDR, is also impaired in heterozygous Brca1/Bard1 mutant cells, SFP and HDR may contribute to distinct stages of tumorigenesis in BRCA1/BARD1 mutation carriers.

Pulmonary administration of curcumin inhibits B16F10 melanoma lung metastasis and invasion in mice.

PURPOSE: Curcumin is expected to have beneficial effects including an anti-cancer effect. However, its lower bioavailability is a critical concern and limits the utility of curcumin in clinical practice. In this study, we investigated whether transpulmonary delivery of curcumin is pharmacologically effective along with improving its bioavailability in mice with lung metastasis. METHODS: C57BL/6J mice were injected with B16F10 melanoma cells via their tail vein and given curcumin by pulmonary administration every other day. The lung tissue of the vehicle-treated mice on day 17 was covered by nodules of metastatic melanoma. RESULTS: Pulmonary curcumin administration significantly and dose-dependently protected the lung metastasis of melanoma. The phosphorylation of JNK (c-Jun NH2 terminal kinase) and HLJ1 expression levels in the lung metastatic nodules of the melanoma were significantly increased by pulmonary curcumin administration. The anti-metastatic effect of curcumin was blunted in mice injected with HLJ1 knocked-down B16F10 melanoma. Systemic bioavailability after pulmonary administration was 61-times higher than after oral administration. Additionally, the curcumin concentration in the lung tissue was sustained to a high level until 24 h after pulmonary administration. CONCLUSIONS: This study showed the usefulness of curcumin to suppress lung metastasis of melanoma by pulmonary administration, a method that may overcome the low-bioavailability of curcumin.

Intranasal administration of neuromedin U derivatives containing cell-penetrating peptides and a penetration-accelerating sequence induced memory improvements in mice.

Neuromedin U (NMU) is a neuropeptide that is expressed and secreted in the brain and gut. We previously demonstrated that the intracerebroventricular (i.c.v.) administration of NMU inhibited inflammation-mediated memory impairment in mice. In order to utilize NMU as a clinical treatment tool for inflammation-mediated amnesia, we herein focused on non-invasive intranasal delivery because the i.c.v. administration route is invasive and impractical. In the present study, we prepared two NMU derivatives containing cell-penetrating peptides (CPPs), octaarginine (R8), and each penetration-accelerating sequence, namely FFLIPKG (PASR8-NMU) and FFFFG (F4R8-NMU), for intranasal (i.n.) administration. In the Y-maze test, the i.c.v. administration of lipopolysaccharide (LPS) (10µg/mouse) significantly decreased spontaneous alternation behavior, and this was prevented by the prior administration of PASR8-NMU or F4R8-NMU (5.6µg/mouse, i.n.). Moreover, the administration of PASR8-NMU or F4R8-NMU (5.6µg/mouse, i.n.) just before the Y-maze test also improved LPS-induced memory impairment. Indocyanine green (ICG)-labeled PASR8-NMU (i.n.) was significantly observed in the hippocampus and paraventricular hypothalamic nucleus 30min after its i.n. administration. PASR8-NMU, but not F4R8-NMU guaranteed the stability of the administration liquid for 24h. These results suggest that PASR8-NMU is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of inflammation-mediated amnesia.

Dosing-time-dependent effect of rivaroxaban on coagulation activity in rats.

The anticoagulant effect of rivaroxaban, a direct inhibitor of activated factor X (FX), might be influenced by its dosing time because the activity of the coagulofibrinolytic system exhibits daily rhythmicity. In rats, FX activity follows a 24-h rhythm with a peak in the middle of the light phase and a trough at the beginning of the dark phase. Consistent with these findings, a single dose of rivaroxaban had a stronger inhibitory effect on FX activity after dosing at the beginning of the light phase than after dosing at the beginning of the dark phase. A similar chronopharmacological effect was seen in a quantitative model of venous stasis thrombosis. In comparison, the dosing time had minimal influence on the pharmacokinetics of rivaroxaban. These data indicate that the anticoagulant effect of rivaroxaban is influenced by the dosing time. Further studies should confirm this finding in a clinical setting.

Effect of 4-[(5,6,7,8-Tetrahydro-5,5,8,8-Tetramethyl-2-Naphthalenyl)Carbamoyl]Benzoic Acid (Am80) on Alveolar Regeneration in Adiponectin Deficient-Mice Showing a Chronic Obstructive Pulmonary Disease-Like Pathophysiology.

Chronic obstructive pulmonary disease (COPD) is an intractable pulmonary disease that causes widespread and irreversible alveolar collapse. Although COPD occurs worldwide, only symptomatic therapy is currently available. Our objective is the development of therapeutic agents to eradicate COPD. Therefore, we focused on 4-[(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) carbamoyl] benzoic acid (Am80), which is a derivative of all-trans retinoic acid. We evaluated the effects of Am80 on alveolar repair in a novel COPD model of adiponectin-deficient mice. This mouse model has more symptoms similar to human COPD than the classic elastase-induced emphysema mouse model. Lung volume, computed tomography (CT) values, low-attenuation area ratios, and bone and fat mass were measured by CT. However, the administration of Am80 did not affect these results. To examine the degree of destruction in the alveoli, the mean linear intercept of the alveolar walls was calculated, and assessment of this value confirmed that there was a significant difference between the control (46.3 ± 2.3 µm) and 0.5 mg/kg Am80-treated group (34.4 ± 1.7 µm). All mice survived the treatment, which lasted for more than 6 months, and we did not observe any abnormalities in autopsies performed at 80 weeks of age. These results suggested that Am80 was effective as a novel therapeutic compound for the treatment of COPD.

Antidepressant-like effects exerted by the intranasal administration of a glucagon-like peptide-2 derivative containing cell-penetrating peptides and a penetration-accelerating sequence in mice.

The intracerebroventicular (i.c.v.) administration of glucagon-like peptide-2 (GLP-2) to rodents was shown to have antidepressant-like effects in imipramine-resistant depression-model mice. In order to utilize GLP-2 as a clinical treatment tool for depression, we herein focused on the intranasal delivery that is non-invasive approach, because the i.c.v. administration is invasive and impractical. In the present study, we prepared a GLP-2 derivative containing cell penetrating peptides (CPPs) and a penetration accelerating sequence (PAS) (PAS-CPPs-GLP-2) for the intranasal (i.n.) administration. PAS-CPPs-GLP-2 (i.n.) exhibited antidepressant-like effects in the forced-swim test (FST) and tail suspension test (TST) in naïve mice as well as adrenocorticotropic hormone (ACTH) treated-mice. However, PAS-CPPs-GLP-2 (i.v.) and the GLP-2 derivative containing CPPs without a PAS (CPPs-GLP-2) (i.n.) did not affect the immobility time in the mouse FST. Moreover, fluorescein isothiocyanate (FITC)-labeled PAS-CPPs-GLP-2 (i.n.), but not FITC-labeled CPPs-GLP-2 (i.n.) was distributed through the mouse brain after the FST session. These results suggest that PAS-CPPs-GLP-2 is effective for i.n. delivery to the brain, and may be useful in the clinical treatment of major depression.

LARETH-25 and ß-CD improve central transitivity and central pharmacological effect of the GLP-2 peptide.

Depression is a common mental disorder. More than 350 million people of all ages suffer from depression worldwide. Although a number of antidepressants are available, >20% of patients with major depressive disorder suffer from treatment-resistant depression. Therefore, development of novel therapeutics to overcome this condition is required. We reported that intracerebroventricular administration of glucagon-like peptide-2 (GLP-2) exerts antidepressant-like effects treated with or without adrenocorticotropic hormone. In the present study, we developed a nasal formulation of GLP-2 containing 5% polyoxyethylene (25) lauryl ether and 1% ß-cyclodextrin that enhanced the resistance of GLP-2 to inactivation by dipeptidyl peptidase-4. Intranasal administration of this formulation (60µg/kg) increased the delivery of GLP-2 to the brain and had antidepressant-like effects on rats. These results suggest the potential of the GLP-2 nasal formulation for use as a novel antidepressant.

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

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 1µg/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 2weeks recovered tissue elastance and forced expiratory volume in 0.05s 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.

Pulmonary Administration of GW0742, a High-Affinity Peroxisome Proliferator-Activated Receptor Agonist, Repairs Collapsed Alveoli in an Elastase-Induced Mouse Model of Emphysema.

Pulmonary emphysema is a disease in which lung alveoli are irreversibly damaged, thus compromising lung function. Our previous study revealed that all-trans-retinoic acid (ATRA) induces the differentiation of human lung alveolar epithelial type 2 progenitor cells and repairs the alveoli of emphysema model mice. ATRA also reportedly has the ability to activate peroxisome proliferator-activated receptor (PPAR) ß/δ. A selective PPARß/δ ligand has been reported to induce the differentiation of human keratinocytes during wound repair. Here, we demonstrate that treatment using a high-affinity PPARß/δ agonist, GW0742, reverses the lung tissue damage induced by elastase in emphysema-model mice and improves respiratory function. Mice treated with elastase, which collapsed their alveoli, were then treated with either 10% dimethyl sulfoxide (DMSO) in saline (control group) or GW0742 (1.0 mg/kg twice a week) by pulmonary administration. Treatment with GW0742 for 2 weeks increased the in vivo expression of surfactant proteins A and D, which are known alveolar type II epithelial cell markers. GW0742 treatment also shortened the average distance between alveolar walls in the lungs of emphysema model mice, compared with a control group treated with 10% DMSO in saline. Treatment with GW0742 for 3 weeks also improved tissue elastance (cm H2O/mL), as well as the ratio of the forced expiratory volume in the first 0.05 s to the forced vital capacity (FEV 0.05/FVC). In each of these experiments, GW0742 treatment reversed the damage caused by elastase. In conclusion, PPARß/δ agonists are potential therapeutic agents for pulmonary emphysema.