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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

Expression of KGF protein in mouse quadriceps muscle and lungs. KGF-FLAG expression in mouse quadriceps muscle was analyzed by Western blotting using an anti-FLAG antibody on day 2 after gene transfection of mice (A). The concentration of KGF in mouse quadriceps muscle (B) and lungs (C) on day 3 and day 7 after gene transfection was measured using an ELISA. * represents p<0.05 compared to pFLAG control.
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Figure 2: Expression of KGF protein in mouse quadriceps muscle and lungs. KGF-FLAG expression in mouse quadriceps muscle was analyzed by Western blotting using an anti-FLAG antibody on day 2 after gene transfection of mice (A). The concentration of KGF in mouse quadriceps muscle (B) and lungs (C) on day 3 and day 7 after gene transfection was measured using an ELISA. * represents p<0.05 compared to pFLAG control.

Mentions: Expression of the KGF-FLAG protein in the quadriceps muscle of electroporated mice was examined by Western blot analysis using an anti-FLAG antibody. A specific band corresponding to KGF-FLAG (28 kDa) was detected on day 2 after pKGF-FLAG gene transfection (Fig. 2A). The human KGF protein levels in the mouse quadriceps and lung after pKGF-FLAG gene transfection were next measured using an ELISA. The levels of KGF protein were 12200±125 pg/mg of tissue weight on day 3 and 1475±327 pg/mg of tissue weight on day 7 in the quadriceps muscles (Fig. 2B), and 287±41.6 pg/mg of tissue weight on day 3 and 137±11.5 pg/mg of tissue weight on day 7 in the lung (Fig. 2C). KGF protein levels in the mice transfected with the pFLAG gene were below measurable limits (<15 pg/ml) and endogenous KGF was not detected in the quadriceps and lung by ELISA. Therefore, changes in the KGF protein level detected by ELISA definitely reflect changes in the levels of the rhKGF protein. rhKGF expression in the quadriceps and the lung had decreased by day 7 after pKGF-FLAG gene transfection compared to day 3 after transfection. These results may indicate that systemic spreading of rhKGF was induced by pKGF-FLAG transfection into the leg muscles of mice.


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)

Expression of KGF protein in mouse quadriceps muscle and lungs. KGF-FLAG expression in mouse quadriceps muscle was analyzed by Western blotting using an anti-FLAG antibody on day 2 after gene transfection of mice (A). The concentration of KGF in mouse quadriceps muscle (B) and lungs (C) on day 3 and day 7 after gene transfection was measured using an ELISA. * represents p<0.05 compared to pFLAG control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4491498&req=5

Figure 2: Expression of KGF protein in mouse quadriceps muscle and lungs. KGF-FLAG expression in mouse quadriceps muscle was analyzed by Western blotting using an anti-FLAG antibody on day 2 after gene transfection of mice (A). The concentration of KGF in mouse quadriceps muscle (B) and lungs (C) on day 3 and day 7 after gene transfection was measured using an ELISA. * represents p<0.05 compared to pFLAG control.
Mentions: Expression of the KGF-FLAG protein in the quadriceps muscle of electroporated mice was examined by Western blot analysis using an anti-FLAG antibody. A specific band corresponding to KGF-FLAG (28 kDa) was detected on day 2 after pKGF-FLAG gene transfection (Fig. 2A). The human KGF protein levels in the mouse quadriceps and lung after pKGF-FLAG gene transfection were next measured using an ELISA. The levels of KGF protein were 12200±125 pg/mg of tissue weight on day 3 and 1475±327 pg/mg of tissue weight on day 7 in the quadriceps muscles (Fig. 2B), and 287±41.6 pg/mg of tissue weight on day 3 and 137±11.5 pg/mg of tissue weight on day 7 in the lung (Fig. 2C). KGF protein levels in the mice transfected with the pFLAG gene were below measurable limits (<15 pg/ml) and endogenous KGF was not detected in the quadriceps and lung by ELISA. Therefore, changes in the KGF protein level detected by ELISA definitely reflect changes in the levels of the rhKGF protein. rhKGF expression in the quadriceps and the lung had decreased by day 7 after pKGF-FLAG gene transfection compared to day 3 after transfection. These results may indicate that systemic spreading of rhKGF was induced by pKGF-FLAG transfection into the leg muscles of mice.

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