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Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.

Zhu P, Yu H, Huang S, Xiang H, Li F, Zheng W - Mol Med Rep (2015)

Bottom Line: The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells.When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently.In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.

ABSTRACT
Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316‑hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription‑quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl‑thiazolyl‑tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently. In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

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Related in: MedlinePlus

Inhibitory effects of Ad-hTK1-hTIMP1 on cell growth and proliferation in VSMCs. (A) Concentration-dependent inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. Cells treated with 0.2% fetal bovine serum were used as a control. *P<0.05 and **P<0.01, compared with cells infected with 20 MOI Ad-hTK1-hTIMP1; #P<0.05 and ##P<0.05, as compared with the cells infected with Ad-EGFP. (B) Time-dependent (1–6 days) inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. *P<0.05, **P<0.01, compared with Ad-hTK1-hTIMP1 for 2 days. (C and D) Inhibitory proliferation effects of co-expression vector evaluated using cell counting and a 3-[4,5-dimethy1-2-thiazolyl]-2,5-dipheny1-2-tetra-zoliumbromide assay. *P<0.01, compared with cells stimulated with platelet-derived growth factor-BB only; ▲P<0.05 as compared with cells infected with Ad-hTK1; ΔP<0.05 as compared with cells infected with Ad-hTIMP1. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1; VSMCs, vascular smooth muscle cells.
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f4-mmr-12-04-5671: Inhibitory effects of Ad-hTK1-hTIMP1 on cell growth and proliferation in VSMCs. (A) Concentration-dependent inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. Cells treated with 0.2% fetal bovine serum were used as a control. *P<0.05 and **P<0.01, compared with cells infected with 20 MOI Ad-hTK1-hTIMP1; #P<0.05 and ##P<0.05, as compared with the cells infected with Ad-EGFP. (B) Time-dependent (1–6 days) inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. *P<0.05, **P<0.01, compared with Ad-hTK1-hTIMP1 for 2 days. (C and D) Inhibitory proliferation effects of co-expression vector evaluated using cell counting and a 3-[4,5-dimethy1-2-thiazolyl]-2,5-dipheny1-2-tetra-zoliumbromide assay. *P<0.01, compared with cells stimulated with platelet-derived growth factor-BB only; ▲P<0.05 as compared with cells infected with Ad-hTK1; ΔP<0.05 as compared with cells infected with Ad-hTIMP1. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1; VSMCs, vascular smooth muscle cells.

Mentions: It was investigated whether the co-expression vector of hTK1 and hTIMP1 genes had an effect on VSMC growth and proliferation. Primary VSMCs were infected with Ad-hTK1-hTIMP1 or Ad-EGFP, or left untreated as a control, and treated with or without PDGF-BB. The cell growth was measured using a cell counting assay. The number of cells induced by PDGF-BB was significantly increased compared with the control. However, Ad-hTK1-hTIMP1 significantly decreased cell numbers as compared with Ad-EGFP (P<0.01). As is shown in Fig. 4A, Ad-hTK1-hTIMP1 had minor inhibitory effects on VSMC proliferation at lower concentrations (<20 MOI). Cell numbers were significantly reduced at concentrations >50 MOI of Ad-hTK1-hTIMP1 (P<0.05) as compared with 20 MOI. Compared with 100 MOI, the inhibition rate was 45.27% when VSMCs were treated with 150 MOI (P<0.05). Cell number of cells infected with 200 MOI of Ad-hTK1-hTIMP1 were no further decreased than those infected with 150 MOI (P>0.05). However, certain cells infected with Ad-hTK1-hTIMP1 at 200 MOI appeared to shrink and float, suggesting apoptosis and necrosis. Therefore, 150 MOI of Ad-hTK1-hTIMP1 was the optimal dose for the present study.


Synergistic effect of a tissue kallikrein 1 and tissue inhibitor of matrix metalloproteinase 1 co‑expression vector on the proliferation of rat vascular smooth muscle cells.

Zhu P, Yu H, Huang S, Xiang H, Li F, Zheng W - Mol Med Rep (2015)

Inhibitory effects of Ad-hTK1-hTIMP1 on cell growth and proliferation in VSMCs. (A) Concentration-dependent inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. Cells treated with 0.2% fetal bovine serum were used as a control. *P<0.05 and **P<0.01, compared with cells infected with 20 MOI Ad-hTK1-hTIMP1; #P<0.05 and ##P<0.05, as compared with the cells infected with Ad-EGFP. (B) Time-dependent (1–6 days) inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. *P<0.05, **P<0.01, compared with Ad-hTK1-hTIMP1 for 2 days. (C and D) Inhibitory proliferation effects of co-expression vector evaluated using cell counting and a 3-[4,5-dimethy1-2-thiazolyl]-2,5-dipheny1-2-tetra-zoliumbromide assay. *P<0.01, compared with cells stimulated with platelet-derived growth factor-BB only; ▲P<0.05 as compared with cells infected with Ad-hTK1; ΔP<0.05 as compared with cells infected with Ad-hTIMP1. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1; VSMCs, vascular smooth muscle cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4581792&req=5

f4-mmr-12-04-5671: Inhibitory effects of Ad-hTK1-hTIMP1 on cell growth and proliferation in VSMCs. (A) Concentration-dependent inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. Cells treated with 0.2% fetal bovine serum were used as a control. *P<0.05 and **P<0.01, compared with cells infected with 20 MOI Ad-hTK1-hTIMP1; #P<0.05 and ##P<0.05, as compared with the cells infected with Ad-EGFP. (B) Time-dependent (1–6 days) inhibitory effects of co-expression vector on VSMC growth assessed by cell counting. *P<0.05, **P<0.01, compared with Ad-hTK1-hTIMP1 for 2 days. (C and D) Inhibitory proliferation effects of co-expression vector evaluated using cell counting and a 3-[4,5-dimethy1-2-thiazolyl]-2,5-dipheny1-2-tetra-zoliumbromide assay. *P<0.01, compared with cells stimulated with platelet-derived growth factor-BB only; ▲P<0.05 as compared with cells infected with Ad-hTK1; ΔP<0.05 as compared with cells infected with Ad-hTIMP1. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1; VSMCs, vascular smooth muscle cells.
Mentions: It was investigated whether the co-expression vector of hTK1 and hTIMP1 genes had an effect on VSMC growth and proliferation. Primary VSMCs were infected with Ad-hTK1-hTIMP1 or Ad-EGFP, or left untreated as a control, and treated with or without PDGF-BB. The cell growth was measured using a cell counting assay. The number of cells induced by PDGF-BB was significantly increased compared with the control. However, Ad-hTK1-hTIMP1 significantly decreased cell numbers as compared with Ad-EGFP (P<0.01). As is shown in Fig. 4A, Ad-hTK1-hTIMP1 had minor inhibitory effects on VSMC proliferation at lower concentrations (<20 MOI). Cell numbers were significantly reduced at concentrations >50 MOI of Ad-hTK1-hTIMP1 (P<0.05) as compared with 20 MOI. Compared with 100 MOI, the inhibition rate was 45.27% when VSMCs were treated with 150 MOI (P<0.05). Cell number of cells infected with 200 MOI of Ad-hTK1-hTIMP1 were no further decreased than those infected with 150 MOI (P>0.05). However, certain cells infected with Ad-hTK1-hTIMP1 at 200 MOI appeared to shrink and float, suggesting apoptosis and necrosis. Therefore, 150 MOI of Ad-hTK1-hTIMP1 was the optimal dose for the present study.

Bottom Line: The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells.When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently.In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

View Article: PubMed Central - PubMed

Affiliation: Department of Geriatrics, Fujian Provincial Hospital Key Laboratory of Geriatrics, Fujian Medical University, Fuzhou, Fujian 350001, P.R. China.

ABSTRACT
Tissue kallikrein 1 (TK1) and tissue inhibitor of matrix metalloproteinase 1 (TIMP1) are important in inhibiting vascular smooth muscle cell (VSMC) proliferation and improving vascular remodeling, respectively. It was hypothesized that a combination of TK1 and TIMP1 genes, mediated by an adenovirus vector could augment or act in synergy to enhance the inhibitory effects. The promoter, mCMV carrying hTIMP1 cDNA was subcloned into pDC316‑hTK1 to construct a recombinant plasmid carrying hTK1 and hTIMP1 genes. Subsequently, the double gene plasmid and adenovirus backbone plasmid were packaged into HEK293A cells. Gene transcription and protein expression were examined, respectively using reverse transcription‑quantitative polymerase chain reaction (PCR) and western blotting assays. VSMC proliferation was assessed using cell counting and methyl‑thiazolyl‑tetrazoliuin methods. The constructed plasmid containing hTK1 and hTIMP1 genes was correctly identified by means of PCR, double digestion and sequencing analysis. The co‑expression vector, Ad‑hTK1‑hTIMP1 was successfully constructed and packaged into HEK293A cells. When VSMCs were transfected with the co‑expression vector, the mRNA transcription and protein expression of hTK1 and hTIMP1 exhibited abundant expression in a concentration‑dependent and time‑dependent manner, independently. In conclusion, the co‑expression vector synergistically inhibited the cell growth and proliferation induced by platelet‑derived growth factor‑BB compared with the single gene vector.

Show MeSH
Related in: MedlinePlus