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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

MOI-dependent co-expression of vascular smooth muscle cells infected with Ad-hTK1-hTIMP1. (A) Results of mRNA expression levels. Reseults are presented as the mean ± standard deviation of 3 independent experiments *P<0.05 and **P<0.01, compared with cells infected with 50 MOI. #P<0.01, compared with mRNA expression of hTIMP1 in cells transferred with 100–150 MOI. (B and C) Expression of hTK protein assessed using western blot analysis. *P<0.05 and **P<0.01, compared with 20 MOI. &P<0.01, compared with 50 MOI. #P<0.01, compared with expression of hTIMP1 protein in cells infected with 50–200 MOI. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
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f3-mmr-12-04-5671: MOI-dependent co-expression of vascular smooth muscle cells infected with Ad-hTK1-hTIMP1. (A) Results of mRNA expression levels. Reseults are presented as the mean ± standard deviation of 3 independent experiments *P<0.05 and **P<0.01, compared with cells infected with 50 MOI. #P<0.01, compared with mRNA expression of hTIMP1 in cells transferred with 100–150 MOI. (B and C) Expression of hTK protein assessed using western blot analysis. *P<0.05 and **P<0.01, compared with 20 MOI. &P<0.01, compared with 50 MOI. #P<0.01, compared with expression of hTIMP1 protein in cells infected with 50–200 MOI. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.

Mentions: VSMCs were infected with Ad-hTK1-hTIMP1 at an MOI of 20, 50, 100, 150 and 200, respectively, and mRNA expression was estimated by indirect RT-qPCR. As is shown in Fig. 3A, MOI-dependent expression was observed at 50–150 MOI at 72 h (P<0.05 50–100 MOI, P<0.01 150 MOI), respectively, whereas there was a lower level of expression in adenovirus-infected cells at 20–50 MOI (P>0.05). However, no significant difference was identified between 150 MOI and 200 MOI (P>0.05).


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)

MOI-dependent co-expression of vascular smooth muscle cells infected with Ad-hTK1-hTIMP1. (A) Results of mRNA expression levels. Reseults are presented as the mean ± standard deviation of 3 independent experiments *P<0.05 and **P<0.01, compared with cells infected with 50 MOI. #P<0.01, compared with mRNA expression of hTIMP1 in cells transferred with 100–150 MOI. (B and C) Expression of hTK protein assessed using western blot analysis. *P<0.05 and **P<0.01, compared with 20 MOI. &P<0.01, compared with 50 MOI. #P<0.01, compared with expression of hTIMP1 protein in cells infected with 50–200 MOI. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3-mmr-12-04-5671: MOI-dependent co-expression of vascular smooth muscle cells infected with Ad-hTK1-hTIMP1. (A) Results of mRNA expression levels. Reseults are presented as the mean ± standard deviation of 3 independent experiments *P<0.05 and **P<0.01, compared with cells infected with 50 MOI. #P<0.01, compared with mRNA expression of hTIMP1 in cells transferred with 100–150 MOI. (B and C) Expression of hTK protein assessed using western blot analysis. *P<0.05 and **P<0.01, compared with 20 MOI. &P<0.01, compared with 50 MOI. #P<0.01, compared with expression of hTIMP1 protein in cells infected with 50–200 MOI. TK1, tissue kallikrein 1; TIMP1, tissue inhibitor of matrix metalloproteinase 1.
Mentions: VSMCs were infected with Ad-hTK1-hTIMP1 at an MOI of 20, 50, 100, 150 and 200, respectively, and mRNA expression was estimated by indirect RT-qPCR. As is shown in Fig. 3A, MOI-dependent expression was observed at 50–150 MOI at 72 h (P<0.05 50–100 MOI, P<0.01 150 MOI), respectively, whereas there was a lower level of expression in adenovirus-infected cells at 20–50 MOI (P>0.05). However, no significant difference was identified between 150 MOI and 200 MOI (P>0.05).

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