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PEP-1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production.

Tan LG, Xiao JH, Yu DL, Zhang L, Zheng F, Guo LY, Yang JY, Tang JM, Chen SY, Wang JN - BMC Cardiovasc Disord (2015)

Bottom Line: However, the effect of copper, zinc-superoxide dismutase (SOD1) as a therapeutic agent is limited due to the insufficient transduction.Protein expression was analyzed by western blotting.PEP-1-SOD1 suppressed MCF proliferation and differentiation and reduced production of collagen type I and III.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, P. R. China. 28621855@qq.com.

ABSTRACT

Background: Oxidative stress is closely associated with cardiac fibrosis. However, the effect of copper, zinc-superoxide dismutase (SOD1) as a therapeutic agent is limited due to the insufficient transduction. This study was aimed to investigate the effect of PEP-1-SOD1 fusion protein on angiotensin II (ANG II)-induced collagen metabolism in rat cardiac myofibroblasts (MCFs).

Methods: MCFs were pretreated with SOD1 or PEP-1-SOD1 fusion protein for 2 h followed by incubation with ANG II for 24 h. Cell proliferation was measured by Cell Counting Kit-8. Superoxide anion productions were detected by both fluorescent microscopy and Flow Cytometry. MMP-1 and TIMP-1 were determined by ELISA. Intracellular MDA content and SOD activity were examined by commercial assay kits. Protein expression was analyzed by western blotting.

Results: PEP-1-SOD1 fusion protein efficiently transduced into MCF, scavenged intracellular O2 (-), decreased intracellular MDA content, increased SOD activity, suppressed ANG II-induced proliferation, reduced expression of TGF-β1, α-SMA, collagen type I and III, restored MMP-1 secretion, and attenuated TIMP-1 secretion.

Conclusion: PEP-1-SOD1 suppressed MCF proliferation and differentiation and reduced production of collagen type I and III. Therefore, PEP-1-SOD1 fusion protein may be a potential novel therapeutic agent for cardiac fibrosis.

No MeSH data available.


Related in: MedlinePlus

Transduction and enzyme activities of PEP-1-SOD1 fusion protein in MCF. a-d: Time and dose-dependent transduction of PEP-1-SOD1. a, b: 2 μM of PEP-1-SOD1 was incubated with cardiac myofibroblast for 0–2 h as indicated, and western blot was performed using anti-His tag antibody (a). b: Semi-quantitative assay was done in figure A. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). c: 0-2 μM of PEP-1-SOD1 was incubated with cells for 2 h. Western blots were performed the same as in A. d: Semi-quantitative assay was done in figure C. #P < 0.01 vs. untreated cells (CTL) group; $P < 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). e: FITC-conjugated PEP-1-SOD1 transduction was detected by immunofluorescent microscopy. f: PEP-1-SOD1 exhibited SOD enzymatic activity in MCF. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 2 h group; $P < 0.05 vs. 4 h group; &P < 0.05 vs. 6 h group; *P < 0.01 vs. 12 h group. (n = 5)
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Fig1: Transduction and enzyme activities of PEP-1-SOD1 fusion protein in MCF. a-d: Time and dose-dependent transduction of PEP-1-SOD1. a, b: 2 μM of PEP-1-SOD1 was incubated with cardiac myofibroblast for 0–2 h as indicated, and western blot was performed using anti-His tag antibody (a). b: Semi-quantitative assay was done in figure A. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). c: 0-2 μM of PEP-1-SOD1 was incubated with cells for 2 h. Western blots were performed the same as in A. d: Semi-quantitative assay was done in figure C. #P < 0.01 vs. untreated cells (CTL) group; $P < 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). e: FITC-conjugated PEP-1-SOD1 transduction was detected by immunofluorescent microscopy. f: PEP-1-SOD1 exhibited SOD enzymatic activity in MCF. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 2 h group; $P < 0.05 vs. 4 h group; &P < 0.05 vs. 6 h group; *P < 0.01 vs. 12 h group. (n = 5)

Mentions: To test the transduction efficiency of PEP-1-SOD1 fusion proteins into cardiac MCF, we used anti-His tag antibody to detect its protein level. As shown in Fig. 1a and c, the fusion protein was observed in MCF 15 min after incubation with 2 μM of PEP-1-SOD1, and the protein level gradually increased when the incubation time increased. However, incubation with SOD1 protein did not result in accumulation of SOD1 in the cells (data not shown). Moreover, the transduction of PEP-1-SOD1 fusion protein, but not SOD1, occurred in a dose-dependent manner (Fig. 1b and d). To further confirm the transduction, PEP-1-SOD1 or SOD1 was conjugated to FITC fluorescein, and the transduction was observed by immunofluorescent microscopy after incubation with the cells. As shown in Fig. 1e, strong green fluorescent signals were observed in cells pretreated with PEP-1-SOD1, but not with SOD1 protein, demonstrating that PEP-1-SOD1, but not SOD1, can enter cardiac myofibroblasts. Importantly, PEP-1-SOD1 is functionally active. As shown in Fig. 1f, SOD1 activity of PEP-1-SOD1 was 2.5-fold higher compared to the untransduced cells, and this high enzyme activity can last for 12–24 h.Fig. 1


PEP-1-SOD1 fusion proteins block cardiac myofibroblast activation and angiotensin II-induced collagen production.

Tan LG, Xiao JH, Yu DL, Zhang L, Zheng F, Guo LY, Yang JY, Tang JM, Chen SY, Wang JN - BMC Cardiovasc Disord (2015)

Transduction and enzyme activities of PEP-1-SOD1 fusion protein in MCF. a-d: Time and dose-dependent transduction of PEP-1-SOD1. a, b: 2 μM of PEP-1-SOD1 was incubated with cardiac myofibroblast for 0–2 h as indicated, and western blot was performed using anti-His tag antibody (a). b: Semi-quantitative assay was done in figure A. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). c: 0-2 μM of PEP-1-SOD1 was incubated with cells for 2 h. Western blots were performed the same as in A. d: Semi-quantitative assay was done in figure C. #P < 0.01 vs. untreated cells (CTL) group; $P < 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). e: FITC-conjugated PEP-1-SOD1 transduction was detected by immunofluorescent microscopy. f: PEP-1-SOD1 exhibited SOD enzymatic activity in MCF. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 2 h group; $P < 0.05 vs. 4 h group; &P < 0.05 vs. 6 h group; *P < 0.01 vs. 12 h group. (n = 5)
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Fig1: Transduction and enzyme activities of PEP-1-SOD1 fusion protein in MCF. a-d: Time and dose-dependent transduction of PEP-1-SOD1. a, b: 2 μM of PEP-1-SOD1 was incubated with cardiac myofibroblast for 0–2 h as indicated, and western blot was performed using anti-His tag antibody (a). b: Semi-quantitative assay was done in figure A. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). c: 0-2 μM of PEP-1-SOD1 was incubated with cells for 2 h. Western blots were performed the same as in A. d: Semi-quantitative assay was done in figure C. #P < 0.01 vs. untreated cells (CTL) group; $P < 0.05 vs. 0.25 h group; &P < 0.01 vs. 0.5 h group; *P < 0.01 vs. 1.0 h group. (n = 5). e: FITC-conjugated PEP-1-SOD1 transduction was detected by immunofluorescent microscopy. f: PEP-1-SOD1 exhibited SOD enzymatic activity in MCF. #P < 0.01 vs. untreated cells (CTL) group; @P > 0.05 vs. 2 h group; $P < 0.05 vs. 4 h group; &P < 0.05 vs. 6 h group; *P < 0.01 vs. 12 h group. (n = 5)
Mentions: To test the transduction efficiency of PEP-1-SOD1 fusion proteins into cardiac MCF, we used anti-His tag antibody to detect its protein level. As shown in Fig. 1a and c, the fusion protein was observed in MCF 15 min after incubation with 2 μM of PEP-1-SOD1, and the protein level gradually increased when the incubation time increased. However, incubation with SOD1 protein did not result in accumulation of SOD1 in the cells (data not shown). Moreover, the transduction of PEP-1-SOD1 fusion protein, but not SOD1, occurred in a dose-dependent manner (Fig. 1b and d). To further confirm the transduction, PEP-1-SOD1 or SOD1 was conjugated to FITC fluorescein, and the transduction was observed by immunofluorescent microscopy after incubation with the cells. As shown in Fig. 1e, strong green fluorescent signals were observed in cells pretreated with PEP-1-SOD1, but not with SOD1 protein, demonstrating that PEP-1-SOD1, but not SOD1, can enter cardiac myofibroblasts. Importantly, PEP-1-SOD1 is functionally active. As shown in Fig. 1f, SOD1 activity of PEP-1-SOD1 was 2.5-fold higher compared to the untransduced cells, and this high enzyme activity can last for 12–24 h.Fig. 1

Bottom Line: However, the effect of copper, zinc-superoxide dismutase (SOD1) as a therapeutic agent is limited due to the insufficient transduction.Protein expression was analyzed by western blotting.PEP-1-SOD1 suppressed MCF proliferation and differentiation and reduced production of collagen type I and III.

View Article: PubMed Central - PubMed

Affiliation: Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, 442000, P. R. China. 28621855@qq.com.

ABSTRACT

Background: Oxidative stress is closely associated with cardiac fibrosis. However, the effect of copper, zinc-superoxide dismutase (SOD1) as a therapeutic agent is limited due to the insufficient transduction. This study was aimed to investigate the effect of PEP-1-SOD1 fusion protein on angiotensin II (ANG II)-induced collagen metabolism in rat cardiac myofibroblasts (MCFs).

Methods: MCFs were pretreated with SOD1 or PEP-1-SOD1 fusion protein for 2 h followed by incubation with ANG II for 24 h. Cell proliferation was measured by Cell Counting Kit-8. Superoxide anion productions were detected by both fluorescent microscopy and Flow Cytometry. MMP-1 and TIMP-1 were determined by ELISA. Intracellular MDA content and SOD activity were examined by commercial assay kits. Protein expression was analyzed by western blotting.

Results: PEP-1-SOD1 fusion protein efficiently transduced into MCF, scavenged intracellular O2 (-), decreased intracellular MDA content, increased SOD activity, suppressed ANG II-induced proliferation, reduced expression of TGF-β1, α-SMA, collagen type I and III, restored MMP-1 secretion, and attenuated TIMP-1 secretion.

Conclusion: PEP-1-SOD1 suppressed MCF proliferation and differentiation and reduced production of collagen type I and III. Therefore, PEP-1-SOD1 fusion protein may be a potential novel therapeutic agent for cardiac fibrosis.

No MeSH data available.


Related in: MedlinePlus