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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 staining of proliferation cell nuclear antigen (PCNA) and surfactant protein A (SP-A) in mouse lung after PPE instillation with or without KGF gene transfection, and double immunohistochemical staining for PCNA and SP-A or KGFR in mouse lung on day 3 after PPE instillation with KGF gene transfection. (A–E): PCNA-positive cells were detected immunohistochemically in the lungs of mice on day 2 (A) and day 3 (C) after PPE instillation with pFLAG gene transfection, and on day 2 (B) and day 3 (D) after PPE instillation with pKGF-FLAG gene transfection. The PCNA labeling index (LI) for day 2, 3 and 7 was calculated (E). (F–H): SP-A expression in the lungs of mice was detected immunohistochemically on day 3 after PPE administration with pFLAG gene transfection (F) or with pKGF-FLAG gene transfection (G). SP-A signal density was measured with an image-analyzer (H). (I, J): Co-localization of PCNA with SP-A (I) or KGFR (J) was immunohistochemically analyzed. PCNA (red) was detected in the nucleus and SP-A (black) was detected in the cytoplasm of alveolar epithelial cells (I). PCNA (red) was detected in the nucleus and KGFR (black) was detected in the cytoplasmic membrane of alveolar epithelial cells (J). Black arrows (PCNA) and open white arrows (SP-A or KGFR) point to cells with co-localized proteins. Magnification: A, B, C, D, F and G (×100), Bar=200 μm; I and J (×200), Bar=200 μm. * represents p<0.05.
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Figure 6: Immunohistochemical staining of proliferation cell nuclear antigen (PCNA) and surfactant protein A (SP-A) in mouse lung after PPE instillation with or without KGF gene transfection, and double immunohistochemical staining for PCNA and SP-A or KGFR in mouse lung on day 3 after PPE instillation with KGF gene transfection. (A–E): PCNA-positive cells were detected immunohistochemically in the lungs of mice on day 2 (A) and day 3 (C) after PPE instillation with pFLAG gene transfection, and on day 2 (B) and day 3 (D) after PPE instillation with pKGF-FLAG gene transfection. The PCNA labeling index (LI) for day 2, 3 and 7 was calculated (E). (F–H): SP-A expression in the lungs of mice was detected immunohistochemically on day 3 after PPE administration with pFLAG gene transfection (F) or with pKGF-FLAG gene transfection (G). SP-A signal density was measured with an image-analyzer (H). (I, J): Co-localization of PCNA with SP-A (I) or KGFR (J) was immunohistochemically analyzed. PCNA (red) was detected in the nucleus and SP-A (black) was detected in the cytoplasm of alveolar epithelial cells (I). PCNA (red) was detected in the nucleus and KGFR (black) was detected in the cytoplasmic membrane of alveolar epithelial cells (J). Black arrows (PCNA) and open white arrows (SP-A or KGFR) point to cells with co-localized proteins. Magnification: A, B, C, D, F and G (×100), Bar=200 μm; I and J (×200), Bar=200 μm. * represents p<0.05.

Mentions: We performed immunohistochemistry for PCNA to assess the effect of pKGF-FLAG gene transfection on the proliferation of lung cells in the mouse emphysema model. The number of PCNA-positive cells in the lung increased on day 2 (LI=11.95%±3.94%) and day 3 (LI=14.60%±4.38%) after PPE instillation in control mice transfected with the pFLAG gene (Fig. 6A and C). When PCNA levels in the lungs of PPE-instilled mice with pKGF-FLAG gene transfection were compared to those of pFLAG gene-transfected PPE-treated mice, the number of PCNA-positive cells was significantly increased on day 2 (LI=13.90%±4.84%), and further increased on day 3 (LI=19.50%±4.52%) (Fig. 6B, D and E). Subsequently, the number of PCNA-positive cells had decreased by day 7 (LI=7.95%±2.19% with pFLAG gene transfection, LI=​8.95%±2.80% with pKGF-FLAG gene transfection). PCNA-positive cells were identified as alveolar epithelial cells, bronchial epithelial cells, endothelial cells and macrophages. To evaluate the effect of the pKGF-FLAG gene in more detail, we performed immunohistochemistry for SP-A, which was used as a marker of alveolar type II epithelial cells. Staining for SP-A was significantly increased on day 3 in the lungs of PPE-instilled mice with pKGF-FLAG gene transfection, compared to that of PPE-instilled mice with pFLAG gene transfection (p=0.002) (Fig. 6F, G and H). When we performed double-staining for PCNA and SP-A or KGFR on day 3 after PPE instillation with pKGF-FLAG gene transfection, colocalization of PCNA and SP-A was detected in alveolar type II cells (Fig. 6I), and colocalization of PCNA and KGFR was detected in alveolar epithelial cells (Fig. 6J). TUNEL staining was also performed to assess apoptosis due to lung injury. As shown in Fig. 7A, B and C, there was no significant difference in the number of apoptotic cells in the emphysematous lung between mice with or without KGF gene at day 3.


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 staining of proliferation cell nuclear antigen (PCNA) and surfactant protein A (SP-A) in mouse lung after PPE instillation with or without KGF gene transfection, and double immunohistochemical staining for PCNA and SP-A or KGFR in mouse lung on day 3 after PPE instillation with KGF gene transfection. (A–E): PCNA-positive cells were detected immunohistochemically in the lungs of mice on day 2 (A) and day 3 (C) after PPE instillation with pFLAG gene transfection, and on day 2 (B) and day 3 (D) after PPE instillation with pKGF-FLAG gene transfection. The PCNA labeling index (LI) for day 2, 3 and 7 was calculated (E). (F–H): SP-A expression in the lungs of mice was detected immunohistochemically on day 3 after PPE administration with pFLAG gene transfection (F) or with pKGF-FLAG gene transfection (G). SP-A signal density was measured with an image-analyzer (H). (I, J): Co-localization of PCNA with SP-A (I) or KGFR (J) was immunohistochemically analyzed. PCNA (red) was detected in the nucleus and SP-A (black) was detected in the cytoplasm of alveolar epithelial cells (I). PCNA (red) was detected in the nucleus and KGFR (black) was detected in the cytoplasmic membrane of alveolar epithelial cells (J). Black arrows (PCNA) and open white arrows (SP-A or KGFR) point to cells with co-localized proteins. Magnification: A, B, C, D, F and G (×100), Bar=200 μm; I and J (×200), Bar=200 μm. * represents p<0.05.
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Figure 6: Immunohistochemical staining of proliferation cell nuclear antigen (PCNA) and surfactant protein A (SP-A) in mouse lung after PPE instillation with or without KGF gene transfection, and double immunohistochemical staining for PCNA and SP-A or KGFR in mouse lung on day 3 after PPE instillation with KGF gene transfection. (A–E): PCNA-positive cells were detected immunohistochemically in the lungs of mice on day 2 (A) and day 3 (C) after PPE instillation with pFLAG gene transfection, and on day 2 (B) and day 3 (D) after PPE instillation with pKGF-FLAG gene transfection. The PCNA labeling index (LI) for day 2, 3 and 7 was calculated (E). (F–H): SP-A expression in the lungs of mice was detected immunohistochemically on day 3 after PPE administration with pFLAG gene transfection (F) or with pKGF-FLAG gene transfection (G). SP-A signal density was measured with an image-analyzer (H). (I, J): Co-localization of PCNA with SP-A (I) or KGFR (J) was immunohistochemically analyzed. PCNA (red) was detected in the nucleus and SP-A (black) was detected in the cytoplasm of alveolar epithelial cells (I). PCNA (red) was detected in the nucleus and KGFR (black) was detected in the cytoplasmic membrane of alveolar epithelial cells (J). Black arrows (PCNA) and open white arrows (SP-A or KGFR) point to cells with co-localized proteins. Magnification: A, B, C, D, F and G (×100), Bar=200 μm; I and J (×200), Bar=200 μm. * represents p<0.05.
Mentions: We performed immunohistochemistry for PCNA to assess the effect of pKGF-FLAG gene transfection on the proliferation of lung cells in the mouse emphysema model. The number of PCNA-positive cells in the lung increased on day 2 (LI=11.95%±3.94%) and day 3 (LI=14.60%±4.38%) after PPE instillation in control mice transfected with the pFLAG gene (Fig. 6A and C). When PCNA levels in the lungs of PPE-instilled mice with pKGF-FLAG gene transfection were compared to those of pFLAG gene-transfected PPE-treated mice, the number of PCNA-positive cells was significantly increased on day 2 (LI=13.90%±4.84%), and further increased on day 3 (LI=19.50%±4.52%) (Fig. 6B, D and E). Subsequently, the number of PCNA-positive cells had decreased by day 7 (LI=7.95%±2.19% with pFLAG gene transfection, LI=​8.95%±2.80% with pKGF-FLAG gene transfection). PCNA-positive cells were identified as alveolar epithelial cells, bronchial epithelial cells, endothelial cells and macrophages. To evaluate the effect of the pKGF-FLAG gene in more detail, we performed immunohistochemistry for SP-A, which was used as a marker of alveolar type II epithelial cells. Staining for SP-A was significantly increased on day 3 in the lungs of PPE-instilled mice with pKGF-FLAG gene transfection, compared to that of PPE-instilled mice with pFLAG gene transfection (p=0.002) (Fig. 6F, G and H). When we performed double-staining for PCNA and SP-A or KGFR on day 3 after PPE instillation with pKGF-FLAG gene transfection, colocalization of PCNA and SP-A was detected in alveolar type II cells (Fig. 6I), and colocalization of PCNA and KGFR was detected in alveolar epithelial cells (Fig. 6J). TUNEL staining was also performed to assess apoptosis due to lung injury. As shown in Fig. 7A, B and C, there was no significant difference in the number of apoptotic cells in the emphysematous lung between mice with or without KGF gene at day 3.

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