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Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

Tobinaga S, Matsumoto K, Nagayasu T, Furukawa K, Abo T, Yamasaki N, Tsuchiya T, Miyazaki T, Koji T - Acta Histochem Cytochem (2015)

Bottom Line: In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells.Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection.Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Surgical Oncology, Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki, Japan.

ABSTRACT
Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

No MeSH data available.


Related in: MedlinePlus

Immunohistochemical localization of KGF and KGFR proteins in mouse lung after PPE instillation with or without KGF gene transfection. KGF protein expression in the mouse lung was immunohistochemically examined in a normal mouse (A), or on day 3 after PPE instillation into a mouse transfected with the pFLAG gene (B) or the pKGF-FLAG gene (C and D). KGF was detected in alveolar cells and bronchial cells. The signal density of KGF in the lungs of PPE-treated mice with KGF-FLAG gene transfection was significantly greater than that of PPE-treated mice with pFLAG gene transfection (G). KGFR protein expression in the mouse lung was immunohistochemically examined on day 3 after PPE instillation with pFLAG gene transfection (E) or with pKGF-FLAG gene transfection (F). KGFR signal density (H) was similar between (E) and (F). Data are representative of 3 mice per group. Magnification: A, B, C, E and F (×100), Bar=100 μm; D (×400), Bar=50 μm. * represents p<0.05.
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Figure 4: Immunohistochemical localization of KGF and KGFR proteins in mouse lung after PPE instillation with or without KGF gene transfection. KGF protein expression in the mouse lung was immunohistochemically examined in a normal mouse (A), or on day 3 after PPE instillation into a mouse transfected with the pFLAG gene (B) or the pKGF-FLAG gene (C and D). KGF was detected in alveolar cells and bronchial cells. The signal density of KGF in the lungs of PPE-treated mice with KGF-FLAG gene transfection was significantly greater than that of PPE-treated mice with pFLAG gene transfection (G). KGFR protein expression in the mouse lung was immunohistochemically examined on day 3 after PPE instillation with pFLAG gene transfection (E) or with pKGF-FLAG gene transfection (F). KGFR signal density (H) was similar between (E) and (F). Data are representative of 3 mice per group. Magnification: A, B, C, E and F (×100), Bar=100 μm; D (×400), Bar=50 μm. * represents p<0.05.

Mentions: We then immunohistochemically analyzed KGF and KGFR levels in the lungs of non-treated mice and PPE-treated mice. Endogenous KGF protein was detected in alveolar and stromal cells in the lungs of non-treated mice (Fig. 4A). In the 3-day PPE-treated lungs of mice whose quadriceps muscles were transfected with pFLAG gene, KGF expression in the lungs was almost equal to that of non-treated mice (Fig. 4B). However, in the 3-day PPE-treated lungs of mice whose quadriceps muscles were transfected with the pKGF-FLAG gene, the KGF signal was greatly increased after transfection compared to that of the FLAG vector control (Fig. 4C and D). Quantitative analysis of KGF signal density using an image-analyzer revealed that the density of KGF in the lung of these pKGF-FLAG gene-transfected mice was significantly increased to twice the level of that in the pFLAG gene-transfected control (pFLAG gene vs. pKGF-FLAG gene; 7.78%±3.37% vs. 15.09%±4.84% (pixel value/mm2), p=0.001) (Fig. 4G). In contrast, expression of the KGFR in the PPE-treated lungs, which was expressed in epithelial cells, did not change significantly between the pFLAG gene- and pKGF-FLAG gene-transfected mice (Fig. 4E, F and H).


Keratinocyte Growth Factor Gene Electroporation into Skeletal Muscle as a Novel Gene Therapeutic Approach for Elastase-Induced Pulmonary Emphysema in Mice.

Tobinaga S, Matsumoto K, Nagayasu T, Furukawa K, Abo T, Yamasaki N, Tsuchiya T, Miyazaki T, Koji T - Acta Histochem Cytochem (2015)

Immunohistochemical localization of KGF and KGFR proteins in mouse lung after PPE instillation with or without KGF gene transfection. KGF protein expression in the mouse lung was immunohistochemically examined in a normal mouse (A), or on day 3 after PPE instillation into a mouse transfected with the pFLAG gene (B) or the pKGF-FLAG gene (C and D). KGF was detected in alveolar cells and bronchial cells. The signal density of KGF in the lungs of PPE-treated mice with KGF-FLAG gene transfection was significantly greater than that of PPE-treated mice with pFLAG gene transfection (G). KGFR protein expression in the mouse lung was immunohistochemically examined on day 3 after PPE instillation with pFLAG gene transfection (E) or with pKGF-FLAG gene transfection (F). KGFR signal density (H) was similar between (E) and (F). Data are representative of 3 mice per group. Magnification: A, B, C, E and F (×100), Bar=100 μm; D (×400), Bar=50 μm. * represents p<0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Immunohistochemical localization of KGF and KGFR proteins in mouse lung after PPE instillation with or without KGF gene transfection. KGF protein expression in the mouse lung was immunohistochemically examined in a normal mouse (A), or on day 3 after PPE instillation into a mouse transfected with the pFLAG gene (B) or the pKGF-FLAG gene (C and D). KGF was detected in alveolar cells and bronchial cells. The signal density of KGF in the lungs of PPE-treated mice with KGF-FLAG gene transfection was significantly greater than that of PPE-treated mice with pFLAG gene transfection (G). KGFR protein expression in the mouse lung was immunohistochemically examined on day 3 after PPE instillation with pFLAG gene transfection (E) or with pKGF-FLAG gene transfection (F). KGFR signal density (H) was similar between (E) and (F). Data are representative of 3 mice per group. Magnification: A, B, C, E and F (×100), Bar=100 μm; D (×400), Bar=50 μm. * represents p<0.05.
Mentions: We then immunohistochemically analyzed KGF and KGFR levels in the lungs of non-treated mice and PPE-treated mice. Endogenous KGF protein was detected in alveolar and stromal cells in the lungs of non-treated mice (Fig. 4A). In the 3-day PPE-treated lungs of mice whose quadriceps muscles were transfected with pFLAG gene, KGF expression in the lungs was almost equal to that of non-treated mice (Fig. 4B). However, in the 3-day PPE-treated lungs of mice whose quadriceps muscles were transfected with the pKGF-FLAG gene, the KGF signal was greatly increased after transfection compared to that of the FLAG vector control (Fig. 4C and D). Quantitative analysis of KGF signal density using an image-analyzer revealed that the density of KGF in the lung of these pKGF-FLAG gene-transfected mice was significantly increased to twice the level of that in the pFLAG gene-transfected control (pFLAG gene vs. pKGF-FLAG gene; 7.78%±3.37% vs. 15.09%±4.84% (pixel value/mm2), p=0.001) (Fig. 4G). In contrast, expression of the KGFR in the PPE-treated lungs, which was expressed in epithelial cells, did not change significantly between the pFLAG gene- and pKGF-FLAG gene-transfected mice (Fig. 4E, F and H).

Bottom Line: In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells.Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection.Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice.

View Article: PubMed Central - PubMed

Affiliation: Division of Surgical Oncology, Department of Translational Medical Sciences, Nagasaki University Graduate School of Biomedical Sciences , Nagasaki, Japan.

ABSTRACT
Pulmonary emphysema is a progressive disease with airspace destruction and an effective therapy is needed. Keratinocyte growth factor (KGF) promotes pulmonary epithelial proliferation and has the potential to induce lung regeneration. The aim of this study was to determine the possibility of using KGF gene therapy for treatment of a mouse emphysema model induced by porcine pancreatic elastase (PPE). Eight-week-old BALB/c male mice treated with intra-tracheal PPE administration were transfected with 80 μg of a recombinant human KGF (rhKGF)-expressing FLAG-CMV14 plasmid (pKGF-FLAG gene), or with the pFLAG gene expressing plasmid as a control, into the quadriceps muscle by electroporation. In the lung, the expression of proliferating cell nuclear antigen (PCNA) was augmented, and surfactant protein A (SP-A) and KGF receptor (KGFR) were co-expressed in PCNA-positive cells. Moreover, endogenous KGF and KGFR gene expression increased significantly by pKGF-FLAG gene transfection. Arterial blood gas analysis revealed that the PaO2 level was not significantly reduced on day 14 after PPE instillation with pKGF-FLAG gene transfection compared to that of normal mice. These results indicated that KGF gene therapy with electroporation stimulated lung epithelial proliferation and protected depression of pulmonary function in a mouse emphysema model, suggesting a possible method of treating pulmonary emphysema.

No MeSH data available.


Related in: MedlinePlus