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A study of the ultrasound-targeted microbubble destruction based triplex-forming oligodexinucleotide delivery system to inhibit tissue factor expression.

Liang W, Zhang W, Zhao S, Li Q, Yang Y, Liang H, Ceng R - Mol Med Rep (2014)

Bottom Line: The in vivo experiments were established in a similar manner to the in vitro experiments, except that TFO or TFO‑M was injected into rats through the tail vein.The transfection efficiency of TFO in the TFO‑M+U group was higher as compared with the TFO‑M and TFO+U group (P<0.01).The protein and mRNA expression of TF in the TFO‑M+U group was significantly decreased both in vitro and in vivo (P<0.01), as compared with the TFO‑M, TFO+U and SSRE groups.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Xinqiao Hospital, The Third Military Medical University, Chongqing 400038, P.R. China.

ABSTRACT
The efficiency of cellular uptake of triplex‑forming oligodexinucleotides (TFO), and the inhibition of tissue factor (TF) is low. The aim of the present study was to improve the absorption of TFO, and increase the inhibition of TF induced by shear stress both in vitro and in vivo, by using an ultrasound‑targeted microbubble destruction (UTMD)‑based delivery system. TFO‑conjugated lipid ultrasonic microbubbles (TFO‑M) were first constructed and characterised. The absorption of TFO was observed by a fluorescence‑based method, and the inhibition of TF by immunofluorescence and quantitative polymerase chain reaction. ECV304 human umbilical vein endothelial cells were subjected to fluid shear stress for 6 h after treatment with TFO conjugated lipid ultrasonic microbubbles without sonication (TFO‑M group); TFO alone; TFO conjugated lipid ultrasonic microbubbles, plus immediate sonication (TFO+U group and TFO‑M+U group); or mock treated with 0.9% NaCl only (SSRE group). The in vivo experiments were established in a similar manner to the in vitro experiments, except that TFO or TFO‑M was injected into rats through the tail vein. Six hours after the preparation of a carotid stenosis model, the rats were humanely sacrificed. The transfection efficiency of TFO in the TFO‑M+U group was higher as compared with the TFO‑M and TFO+U group (P<0.01). The protein and mRNA expression of TF in the TFO‑M+U group was significantly decreased both in vitro and in vivo (P<0.01), as compared with the TFO‑M, TFO+U and SSRE groups. The UTMD‑based TFO delivery system promoted the -absorption of TFO and the inhibition of TF, and was therefore considered to be favorable for preventing thrombosis induced by shear stress.

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Immunofluorescence assay of tissue factor protein in vivo. (A) In vivo expression of tissue factor protein was detected by immunofluorescence microscopy (magnification, ×400). The arrows indicate positive immunofluorescence of tissue factor protein in endothelial cells of the carotid artery. (B) Average gray value in the SSRE, TFO-M, TFO+U and TFO-M+U groups. The values represent the means ± standard deviation, n=6 per group. #P<0.01 as compared with the SSRE group; *P<0.01 as compared with the TFO-M+U group. TFO, triplex-forming oligonucleotides; M, microbubble; U, ultrasound; SSRE, shear stress response element.
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f6-mmr-11-02-0903: Immunofluorescence assay of tissue factor protein in vivo. (A) In vivo expression of tissue factor protein was detected by immunofluorescence microscopy (magnification, ×400). The arrows indicate positive immunofluorescence of tissue factor protein in endothelial cells of the carotid artery. (B) Average gray value in the SSRE, TFO-M, TFO+U and TFO-M+U groups. The values represent the means ± standard deviation, n=6 per group. #P<0.01 as compared with the SSRE group; *P<0.01 as compared with the TFO-M+U group. TFO, triplex-forming oligonucleotides; M, microbubble; U, ultrasound; SSRE, shear stress response element.

Mentions: A rat model of carotid stenosis was successfully generated. The expression of TF protein in endothelial cells of carotid arteries in the four different groups was detected by immunofluorescence (Fig. 6A). The number of positive cells and the degree of staining was significant in the SSRE group, as compared with the other groups. The amount of red fluorescence in the TFO+U, TFO-M and the TFO-M+U group was lower; with the amount of fluorescence in the TFO-M+U group being significantly lower as compared with the TFO+U and TFO-M groups. Image analysis identified that the TF protein content in the TFO+U (51.22±5.69), TFO-M (55.22±6.47) and the TFO-M+U groups (20.59±4.38) was significantly lower (P<0.01) as compared with the SSRE group (71.78±7.10) (Fig. 6B). The fluorescence in the TFO-M+U group was significantly lower as compared with the TFO+U and TFO-M groups (P<0.01) and there was no significant difference between the TFO+U and TFO-M groups.


A study of the ultrasound-targeted microbubble destruction based triplex-forming oligodexinucleotide delivery system to inhibit tissue factor expression.

Liang W, Zhang W, Zhao S, Li Q, Yang Y, Liang H, Ceng R - Mol Med Rep (2014)

Immunofluorescence assay of tissue factor protein in vivo. (A) In vivo expression of tissue factor protein was detected by immunofluorescence microscopy (magnification, ×400). The arrows indicate positive immunofluorescence of tissue factor protein in endothelial cells of the carotid artery. (B) Average gray value in the SSRE, TFO-M, TFO+U and TFO-M+U groups. The values represent the means ± standard deviation, n=6 per group. #P<0.01 as compared with the SSRE group; *P<0.01 as compared with the TFO-M+U group. TFO, triplex-forming oligonucleotides; M, microbubble; U, ultrasound; SSRE, shear stress response element.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4262506&req=5

f6-mmr-11-02-0903: Immunofluorescence assay of tissue factor protein in vivo. (A) In vivo expression of tissue factor protein was detected by immunofluorescence microscopy (magnification, ×400). The arrows indicate positive immunofluorescence of tissue factor protein in endothelial cells of the carotid artery. (B) Average gray value in the SSRE, TFO-M, TFO+U and TFO-M+U groups. The values represent the means ± standard deviation, n=6 per group. #P<0.01 as compared with the SSRE group; *P<0.01 as compared with the TFO-M+U group. TFO, triplex-forming oligonucleotides; M, microbubble; U, ultrasound; SSRE, shear stress response element.
Mentions: A rat model of carotid stenosis was successfully generated. The expression of TF protein in endothelial cells of carotid arteries in the four different groups was detected by immunofluorescence (Fig. 6A). The number of positive cells and the degree of staining was significant in the SSRE group, as compared with the other groups. The amount of red fluorescence in the TFO+U, TFO-M and the TFO-M+U group was lower; with the amount of fluorescence in the TFO-M+U group being significantly lower as compared with the TFO+U and TFO-M groups. Image analysis identified that the TF protein content in the TFO+U (51.22±5.69), TFO-M (55.22±6.47) and the TFO-M+U groups (20.59±4.38) was significantly lower (P<0.01) as compared with the SSRE group (71.78±7.10) (Fig. 6B). The fluorescence in the TFO-M+U group was significantly lower as compared with the TFO+U and TFO-M groups (P<0.01) and there was no significant difference between the TFO+U and TFO-M groups.

Bottom Line: The in vivo experiments were established in a similar manner to the in vitro experiments, except that TFO or TFO‑M was injected into rats through the tail vein.The transfection efficiency of TFO in the TFO‑M+U group was higher as compared with the TFO‑M and TFO+U group (P<0.01).The protein and mRNA expression of TF in the TFO‑M+U group was significantly decreased both in vitro and in vivo (P<0.01), as compared with the TFO‑M, TFO+U and SSRE groups.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, Xinqiao Hospital, The Third Military Medical University, Chongqing 400038, P.R. China.

ABSTRACT
The efficiency of cellular uptake of triplex‑forming oligodexinucleotides (TFO), and the inhibition of tissue factor (TF) is low. The aim of the present study was to improve the absorption of TFO, and increase the inhibition of TF induced by shear stress both in vitro and in vivo, by using an ultrasound‑targeted microbubble destruction (UTMD)‑based delivery system. TFO‑conjugated lipid ultrasonic microbubbles (TFO‑M) were first constructed and characterised. The absorption of TFO was observed by a fluorescence‑based method, and the inhibition of TF by immunofluorescence and quantitative polymerase chain reaction. ECV304 human umbilical vein endothelial cells were subjected to fluid shear stress for 6 h after treatment with TFO conjugated lipid ultrasonic microbubbles without sonication (TFO‑M group); TFO alone; TFO conjugated lipid ultrasonic microbubbles, plus immediate sonication (TFO+U group and TFO‑M+U group); or mock treated with 0.9% NaCl only (SSRE group). The in vivo experiments were established in a similar manner to the in vitro experiments, except that TFO or TFO‑M was injected into rats through the tail vein. Six hours after the preparation of a carotid stenosis model, the rats were humanely sacrificed. The transfection efficiency of TFO in the TFO‑M+U group was higher as compared with the TFO‑M and TFO+U group (P<0.01). The protein and mRNA expression of TF in the TFO‑M+U group was significantly decreased both in vitro and in vivo (P<0.01), as compared with the TFO‑M, TFO+U and SSRE groups. The UTMD‑based TFO delivery system promoted the -absorption of TFO and the inhibition of TF, and was therefore considered to be favorable for preventing thrombosis induced by shear stress.

Show MeSH
Related in: MedlinePlus