Polymer-based delivering of shRNA to rabbit aortic smooth muscle cells suppressed the expression of IGF-1R in vitro and in vivo.

Restenosis is one of clinical limitations for vein graft in coronary bypass graft. It has been proved that signal pathway IGF-1 and its receptor (IGF-1R) activated by hemodynamic mechanical stretch are responsible for the vascular smooth muscle cells proliferation in vein graft neointima formation. Unfortunately, there is no routinely successful method to resolve this problem. Gene delivering to vein graft possesses great therapeutic potential to prevent neointima formation. Polymer is one kind of nanoparticles, which can activate the process of endocytosis of cells. In this study, we evaluated the transfection efficiency and therapeutic potential of polymer-based transfection of plasmids expressing GFP and shRNAs targeting IGF-1R (pGFPshIGF-1Rs) to smooth muscle cells and rabbit external jugular vein graft. Results showed that polymer-based transfection provided high efficiency of transgene expression in smooth muscle cells in vitro. In vitro, IGF-1R-specific shRNA transfected by polymer inhibited IGF-1R protein expression by 52 ± 3.6%, when compared with mock transfected cells. In vivo delivering efficiency of pGFPshIGF-1R plasmid into the rabbit external jugular vein graft was significantly high in the polymer-based transfection group, when compared with negative control group. In vivo, polymer-based transfection IGF-1R-specific shRNA efficiently inhibited the expression of IGF-1R protein by 77 ± 3.6%, 65.6 ± 4.9%, and 76.7 ± 4.3% at 24, 48, and 72 h, respectively, when compared with negative control group. Our findings indicated that polymer-based transfection may be a promising technique that allows the targeting of gene therapy for vein graft restenosis.

[Magnetic liposome mediated shRNA specifically suppresses the growth of non-small cell lung cancer in vitro and in vivo].

OBJECTIVE: To evaluate the inhibition of shRNA mediated by magnetic liposome in the growth of non-small cell lung cancer (NSCLC) under the interference of magnetic field in vitro and in vivo and explore the effects of magnetic field on the efficiency of magnetofection. METHODS: The plasmid of pGFPshIGF-1R was constructed for expressing GFP and shRNA against IGF-1R. CombiMAG as superparamagnetic iron oxide nanoparticles (SPIONs) and Lipofectamine2000 as cationic liposome comprised the magnetic liposome. pGFPshIGF-1R was transferred into A549 cells by magnetofection under a series of interaction durations and intensity of external magnetic fields. pGFPshIGF-1R was delivered into A549 cells in vitro and injected intravenously into the tumor-bearing mice every 48 h for four doses in vivo by way of lipofection or magnetofection. The magnetofection efficiency was analyzed by cytometry and the potency of IGF-1R knockdown by Western blot. At Week 3 after the 4th injection, the mice were sacrificed and the tumors removed and weighed. The tumor inhibition rate was calculated. RESULTS: The interaction durations and intensity of magnetic field could influence the magnetofection efficiency. In vitro, IGF-1R specific-shRNA transfected by lipofection inhibited IGF-1R protein by 56.1% ± 6.0% and by liposomal magnetofection by 85.1% ± 3.0%. In vivo, pGFPshIGF-1R delivered by both lipofection and magnetofection significantly inhibited the tumor growth by 41.3% (P < 0.01) and 65.2% (P < 0.01). CONCLUSIONS: Based on magnetic liposome as gene vectors, magnetofection may become a promising targeted therapy for lung cancer. And the transfection efficiency is influenced by magnetic field.

[Observation of the antithrombotic property of a new prosthetic heart valve in sheep].

UNLABELLED: To observe the antithrambotic property of a new prosthetic made double bileaflet valve of all-pyrolytic carbon. METHOD: Fifteen sheeps underwent mitral valve replacement (MVR) with this new mechanical valve. Each animal was placed on cardiopulmonary bypass. All of the sheep were given in an anticoagulation protocol and followed for three months. Final studies were performed on all animals surviving for more than 12 weeks. RESULT: Ten sheeps died within 48 h after operation, five sheeps survived. The long-term survivors were killed forautopsies, two sheeps were killed in the 1st week after operation, one in 12th weeks and two in 2.5 years. Pathological examination of 10 sheeps which died early after operation revealed that there was no either evidence of thromboembotic phenomenon, or any failures of the mechanical valve. Autopsy studies in the five long-term survivors revealed that the sewing ring was covered by a thin layer of microthrombi consisted most of platelets and fibrin in 1th week after operation. The sewing rings was covered by a tissue layer, with partial organized micro thrombi and endothelialization in 12th weeks after the operation. Two and half years after operation, the complete endothelialization presented around the sewing ring without restriction of bileaflet motion. The tissue ingrowth tends to stop at the pyrolytic carbon-fabric interface on both surfaces of left atrium and ventricle. This study demonstrated that the new prosthetic heart valve had not promoted thrombosis and thrombembolism.

[Assessing the transvalvular gradient of a Chinese-made jiuling solid pyrolytic carbon bileaflet heart valve prosthesis].

In order to assess the transvalvular gradient of a Chinese-made Jiuling solid pyrolytic carbon bileaflet heart valve prosthesis, we determined its hydrodynamics in the laboratory and then measured the hemodynamic performance in the animals and patiens. The Jiuling prosthesis was tested in a pulsatile flow simulator in the aortic position. Six sheep subjected to mitral replacement with 21 mm Jiuling prosthesis were measured by open cardiac catheterization intraoperatively. Doppler echocardiography and open cardiac catheterization under dobutamine stress were performed in two sheep 60 months after implantation. Clinically, 14 cases of aortic valve and 10 cases of mitral valve in the patients who underwent valve replacement with Jiuling heart valve prosthesis were measured by open cardiac catheterization and Doppler echocardiopgraphy. The results showed that Juiling heart valve prosthesis had lower mean transvalvular gradient (below 10 mmHg) at any given tissue annulus diameter. In animal experiments, the transvalvular gradients of 6 sheep were 5.2 +/- 1.7 mmHg intraoperatively, and of 2 sheep 60 months after implantation were 6.1 +/- 0.3 mmHg by catheterization. In patients, the mean transvalvular gradients in the aortic position measured by means of catheterization and echocardiography were 6.26-4.10 mmHg and 9.42-7.48 mmHg; the gradients in the mitral position were 2.10-1.9 mmHg and 5.281-4.10 mmHg respectively. The above results demonstrate that Jiuling valve prosthesis has excellent hemodynamic performance.

Cardiac-targeting magnetic lipoplex delivery of SH-IGF1R plasmid attenuate norepinephrine-induced cardiac hypertrophy in murine heart.

Recent studies have demonstrated a number of molecular mechanisms contributing to the initiation of cardiac hypertrophy response to pressure overload. IGF1R (insulin-like growth factor-1 receptor), an important oncogene, is overexpressed in hypertrophic heart and mediates the hypertrophic pathology process. In this study, we applied with liposomal magnetofection that potentiated gene transfection by applying an external magnetic field to enhance its transfection efficiency. Liposomal magnetofection provided high efficiency in transgene expression in vivo. In vivo, IGF1R-specific-shRNA (small-hairpin RNA) by magnetofection inhibited IGF1R protein expression by 72.2 ± 6.8, 80.7 ± 9.6 and 84.5 ± 5.6%, at 24, 48 and 72 h, respectively, after pGFPshIGF1R injection, indicating that liposomal magnetofection is a promising method that allows the targeting of gene therapy for heart failure. Furthermore, we found that the treated animals (liposomal magnetofection with shIGF1R) showed reduced septal and posterior wall thickness, reduced HW:BWs (heart weight-to-body weights) compared with controls. Moreover, we also found that liposomal magnetofection-based shIGF1R transfection decreased the expression level of p-ERK (phosphorylated extracellular-signal-regulated kinase)1/2, p-AKT1 (phosphorylated protein kinase B1) compared with untreated hearts. These results suggested that liposomal magnetofection-mediated IGF1R-specific-shRNA may be a promising method, and suppression the IGF1R expression inhibited norepinephrine-induced cardiac hypertrophic process via inhibiting PI3K (phosphoinositide 3-kinase)/AKT pathway.