About this Journal Submit a Manuscript Table of Contents
Journal of Biomedicine and Biotechnology
Volume 2011 (2011), Article ID 839653, 4 pages
http://dx.doi.org/10.1155/2011/839653
Research Article

Protein Expression of Mesenchymal Stem Cells after Transfection of pcDNA3.1-hVEGF165 by Ultrasound-Targeted Microbubble Destruction

1Department of Ultrasound, Second Affiliated Hospital, Zhejiang University College of Medicine, 88 Jiefang Road, Hangzhou 310009, China
2Department of Cardiology, Second Affiliated Hospital, Zhejiang University College of Medicine, 88 Jiefang Road, Hangzhou 310009, China
3Department of Nuclear Medicine, Second Affiliated Hospital, Zhejiang University College of Medicine, 88 Jiefang Road, Hangzhou 310009, China

Received 29 December 2010; Revised 19 March 2011; Accepted 22 March 2011

Academic Editor: Mei Tian

Copyright © 2011 Zhaoxia Pu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Ultrasound-targeted microbubble destruction (UTMD) has been proposed as a new technique for organ-specific gene transfer and drug delivery. This study was performed to investigate the effect of UTMD on marrow mesenchymal stem cells (MSCs) transfected with pcDNA3.1-hVEGF165.pcDNA3.1-hVEGF165 were transfected into the third passage of MSCs, with or without UTMD under different ultrasound conditions. Protein expression was quantified by hVEGF165-ELISA kit after transfection for 24, 48, and 72 hours. UTMD-mediated transfection of MSCs yielded a significant protein expression. UTMD of mechanic index (MI) 0.6 for 90 seconds led to the highest level of protein expression.

1. Introduction

Heart disease currently remains the leading cause of death worldwide. With the development of tissue engineering, stem cell technology has been widely used and highlights the latest advances in these exciting fields [1]. Mesenchymal stem cells (MSCs) have demonstrated the ability to differentiate into cardiomyocytes, but are still limited to construct the vessels [2, 3]. Vascular endothelial growth factor (VEGF) could induce vascular endothelial cell proliferation and angiogenesis [4]. Because of its short half-life, VEGF could not maintain effective concentration in blood after injection [5]. In recent years, Ultrasound-targeted microbubble destruction (UTMD) has been proved to be a promising technique for organ-specific gene and drug delivery [6]. In this experiment, we transferred pcDNA3.1-hVEGF165 into MSCs by UTMD and observed the effect of the protein expression.

2. Materials and Methods

2.1. Separation and Cultivation of MSCs

Our experiment was performed in the Clinical Research Center, the Second Affiliated Hospital, School of Medicine, Zhejiang University, China. Five male Sprague-Dawley rats, weighing 80–100 g, were provided by the animal center of Zhejiang University. All experiments have adhered to the National Institutes of Health guide for the care and use of laboratory animals (NIH Publications no. 8023, revised 1978). Approval from the Institutional Animal Care and Use Committee at Zhejiang University Health Science Centre was also obtained to perform the described experiments. MSCs were harvested from the bone marrow of femurs of these rats. Briefly, bone marrow cells were flushed out with 30 mL complete Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) containing 10% heat-inactivated fetal bovine serum (FBS, Gibco, USA), 5 mg/mL glutamine (Gibco, USA), 100 U/mL penicillin (Gibco, USA), and 100 U/mL streptomycin (Gibco, USA). The cells were grown in a humidified atmosphere containing 5% CO2 and 95% O2. The medium was replaced 24 hours later and refreshed every 2 days. Cells were subcultured according to 1 : 2 ratio when they reached approximately 80% confluence by trypsinization (0.25% trypsin, Gibco, USA). The third passage of MSCs was adopted for transfection.

2.2. Recombinant pcDNA3.1-hVEGF165 Gene Transfer into MSCs by UTMD

The third passage of MSCs were planted into three 6-well plate (Becton Dickinson, USA) at 1.0 × 105 cells/per well and cultured for 24 hours in 37°C, 5% CO2 conditions. Before transfection, 5 mL normal saline were added to the microbubble contrast agent SonoVue (Bracco, Italy) powder (25 mg) and thoroughly mixed for 20 seconds. 4 μg/per well pcDNA3.1-hVEGF165 recombinant (Future Biotech, China) were mixed with 10 mL lipofectamin 2000 transfection reagent (Invitrogen, USA) for 20 s [7].

In this study, all the cells were divided intothe following five groups:(1)the blank control group: MSCs with culture fluid,(2)the control group A: 4 μg pcDNA3.1-hVEGF165 recombinant were transfected into MSCs,(3)the control group B: 4 μg pcDNA3.1-hVEGF165 recombinant mixed with 300 μl SonoVue microbubble were transfected into MSCs,(4)the control group C: 4 μg pcDNA3.1-hVEGF165 recombinant were transfected into MSCs by ultrasonic exposure (illustrated by the example of mechanic index (MI) 1.0 and exposure time (ET) 60 s),(5)the UTMD group: 4 μg pcDNA3.1-hVEGF165 recombinant were transfected into MSCs by UTMD (MI 1.0, ET 60 s).

The UTMD group was also divided into three groups according to different MI and ET.

Ultrasound-targeted microbubble was ruptured as following: Acuson Sequoia 512 ultrasound’s 3V2C transducer (Siemens, German) was placed on the bottom of each well plate according to the preset ultrasonic exposure condition. The ultrasound parameters were set as follows: the frequency was 4 MHz, the depth was 4 cm, MI was 0.6, 1.0, and 1.4 respectively, and ET was 30 s, 60 s, and 90 s, respectively.

MSCs cultural supernatant was collected after transfection for 24, 48, and 72 hours, respectively. Five samples were applied in each group.

2.3. Detection of VEGF165 Protein Expression after Transfection by ELISA Quantitative Assay

hVEGF165-ELISA kit (Jingmei, China) was used to determine VEGF165 protein expression after transfection for 24, 48, and 72 hours according to the instructions.This was repeatedfive times in this experiment.

2.4. Statistical Analysis

All the parameters were expressed as mean ± standard deviation. A one-way analysis of variance (ANOVA), followed by a LSD (least significant difference) test was used to compare VEGF165 protein expression among different groups. All analyses were performed using SPSS statistical software, version 13.0 (SPSS, Inc., USA). A two-sided 𝑃 < . 0 5 was considered statistically significant.

3. Results

The results showed that the VEGF165 protein expression increased at 24 hours and reached the maximum level at 48 hours, then decreased at 72 hours (Table 1). Compared with the control group, protein expression of the UTMD group was significantly increased ( 𝑃 < . 0 5 ) .

tab1
Table 1: Protein expression of VEGF165 in mesenchymal stem cells supernatant after transfection ( 𝑛 = 2 5 , ng/mL).

Table 2 also demonstrated that VEGF165 protein level varied according to different ultrasound conditions. The group with ET 90 s and MI 0.6 showed the highest protein level at 48 hours, which has statistical significance compared with every group with ET 30 s and MI 0.6, 1.0, and 1.4, respectively ( 𝑃 < . 0 5 ) .

tab2
Table 2: Protein expression of VEGF165 in mesenchymal stem cells supernatant after transfection under different ultrasound conditions ( 𝑛 = 2 5 , ng/mL).

4. Discussion

The lack of suitable autologous grafts has produced a need for artificial grafts, but the patency of such grafts is limited compared to natural materials. Tissue engineering, whereby living tissue replacements can be constructed, has emerged as a solution to some of these difficulties [8]. MSCs have demonstrated the ability to differentiate into cardiomyocytes, This, in turn, is limited by the availability of MSCs to construct the vessels [9].

VEGF, a class of molecular weight of 34~45 KD glycoprotein, could induce vascular endothelial cell proliferation and angiogenesis. VEGF165 protein-induced differentiation of MSCs directional vascular endothelial cells plays a vital role in neovascularization of ischemic tissues [10, 11]. However, because of its short half-life, VEGF could not maintain effective concentration in blood after injection because of rapid degradation of deoxyribonucleic acid (DNA) [1214]. Thus, intravenous injection of plasmid DNA does not lead to detectable transfection [15]. In the present study, UTMD, a promising technique for organ-specific gene and drug delivery, was tried aiming to transfer VEGF into MSCs efficiently.

UTMD has evolved as a promising tool for organ-specific gene and drug delivery [16]. This technique has initially been developed as a method in myocardial contrast echocardiography, destroying intramyocardial microbubbles to characterize refill kinetics. When loading similar microbubbles with a bioactive substance, ultrasonic destruction of microbubbles may release the transported substance in the targeted organ [17]. Furthermore, high-amplitude oscillations of microbubbles increased capillary and cell membrane permeability and facilitated tissue and cell penetration of the released substance [1820].

As the target cell of gene transfer, MSCs could promote expression of VEGF protein and vascularization of tissue engineering bone by transfected VEGF165. VEGF165 was a kind of secretary protein, whether the transfected gene could express effectively was the critical point of the present experiment.

Table 1 showed that VEGF165 protein production increased after MSCs was transfected with VEGF165 by UTMD. The VEGF165 protein expression reached maximum at 48 hours and decreased later, which had statistical significance compared with all other non-UTMD group at all set moments ( 𝑃 < . 0 5 ) . It could be explained by three mechanisms: firstly, electron microscopy has demonstrated pore formation on cell membranes immediately after destruction of microbubbles, the pores are transient and disappeared after 24 hours [21]. Such “sonoporation” effects may help facilitating gene or drug entry into the cell. Studies on single bubbles in vitro have shown that even linear bubble oscillations are sufficient to achieve rupture of lipid membranes [22]. Secondly, sudden violent collapse of microbubbles (inertial cavitation) can produce high-velocity fluid microjets that may penetrate adjacent membranes [23]. Thirdly, inertial cavitation, which is dependent on microbubble shell composition, ultrasound frequency, pulse duration, and acoustic power, can lead to secondary shock waves, transient local high temperatures, and shear stress, all of which could potentially contribute to gene or drug delivery by UTMD [24, 25].

Table 2 showed that VEGF165 protein level changed under different ultrasound conditions. The group with UTMD of MI 0.6 for 90 s showed the highest peak protein level at 48 hours, which has statistical significance compared with other groups with ET 30 s. Studies have confirmed that the disruption force of microbubbles is greater when the ultrasound frequency used matches the resonant frequency of microbubbles. Even low acoustic pressures can result in microbubble destruction, but higher pressures will lead to more forceful reactions [26]. However, too higher acoustic pressure will hurt the cells, this is why the VEGF165 protein level of groups with MI 1.4, ET 90 s was lower in this study.

5. Limitations

The first limitation of this present study is that the number of samples is small. However, even with this small number of samples, we were able to reach our primary goal of investigating the protein expression of UTMD on MSCs transfected with pcDNA3.1-hVEGF165. Secondly, the cell proliferation and angiogenesis of transfected MSCs by UTMD will not be traced, which is very important for tissue engineering. Thirdly, this study is limited in vitro. So further investigation, especially in larger animal models, is needed.

6. Conclusion

UTMD-mediated transfection of MSCs yielded a significant protein expression. UTMD of mechanic index (MI) 0.6 for 90 seconds led to the highest level of protein expression.

Acknowledgment

This study was supported by the Health Bureau of Zhejiang Provincial (2010KYB056).

References

  1. K. M. Sales, H. J. Salacinski, N. Alobaid, M. Mikhail, V. Balakrishnan, and A. M. Seifalian, “Advancing vascular tissue engineering: the role of stem cell technology,” Trends in Biotechnology, vol. 23, no. 9, pp. 461–467, 2005. View at Publisher · View at Google Scholar · View at Scopus
  2. J. M. Hare and S. V. Chaparro, “Cardiac regeneration and stem cell therapy,” Current Opinion in Organ Transplantation, vol. 13, no. 5, pp. 536–542, 2008. View at Publisher · View at Google Scholar · View at Scopus
  3. A. Behfar, R. S. Faustino, D. K. Arrell, P. P. Dzeja, C. Perez-Terzic, and A. Terzic, “Guided stem cell cardiopoiesis: discovery and translation,” Journal of Molecular and Cellular Cardiology, vol. 45, no. 4, pp. 523–529, 2008. View at Publisher · View at Google Scholar · View at Scopus
  4. H. Gerhardt, “VEGF and endothelial guidance in angiogenic sprouting,” Organogenesis, vol. 4, no. 4, pp. 241–246, 2008. View at Scopus
  5. P. Douvaras, D. G. Antonatos, K. Kekou et al., “Association of VEGF gene polymorphisms with the development of heart failure in patients after myocardial infarction,” Cardiology, vol. 114, no. 1, pp. 11–18, 2009. View at Publisher · View at Google Scholar · View at Scopus
  6. R. Bekeredjian, P. A. Grayburn, and R. V. Shohet, “Use of ultrasound contrast agents for gene or drug delivery in cardiovascular medicine,” Journal of the American College of Cardiology, vol. 45, no. 3, pp. 329–335, 2005. View at Publisher · View at Google Scholar · View at Scopus
  7. T. Susa, T. Kato, and Y. Kato, “Reproducible transfection in the presence of carrier DNA using FuGENE6 and Lipofectamine 2000,” Molecular Biology Reports, vol. 35, no. 3, pp. 313–319, 2008. View at Publisher · View at Google Scholar · View at Scopus
  8. T. Shinoka and C. Breuer, “Tissue-engineered blood vessels in pediatric cardiac surgery,” Yale Journal of Biology and Medicine, vol. 81, no. 4, pp. 161–166, 2008. View at Scopus
  9. M. Siepe, P. Akhyari, A. Lichtenberg, C. Schlensak, and F. Beyersdorf, “Stem cells used for cardiovascular tissue engineering,” European Journal of Cardio-Thoracic Surgery, vol. 34, no. 2, pp. 242–247, 2008. View at Publisher · View at Google Scholar · View at Scopus
  10. J. Jiang, C. Y. Fan, and B. F. Zeng, “Osteogenic differentiation effects on rat bone marrow-derived mesenchymal stromal cells by lentivirus-mediated co-transfection of human BMP2 gene and VEGF165 gene,” Biotechnology Letters, vol. 30, no. 2, pp. 197–203, 2008. View at Publisher · View at Google Scholar · View at Scopus
  11. C. Q. Gao, M. Yang, L. B. Li et al., “The experimental studies on cell transplantation into chronic ischemic myocardium using mesenchymal stem cells modified by recombinant adenovirus carrying vascular endothelial growth factors 165 gene,” Zhonghua Wai Ke Za Zhi, vol. 45, no. 14, pp. 990–993, 2007. View at Scopus
  12. J. J. Haigh, “Role of VEGF in organogenesis,” Organogenesis, vol. 4, no. 4, pp. 247–256, 2008. View at Scopus
  13. P. E. Pestryakov and O. I. Lavrik, “Mechanisms of single-stranded DNA-binding protein functioning in cellular DNA metabolism,” Biochemistry, vol. 73, no. 13, pp. 1388–1404, 2008. View at Publisher · View at Google Scholar · View at Scopus
  14. K. Kawabata, Y. Takakura, and M. Hashida, “The fate of plasmid DNA after intravenous injection in mice: involvement of scavenger receptors in its hepatic uptake,” Pharmaceutical Research, vol. 12, no. 6, pp. 825–830, 1995. View at Scopus
  15. V. T. G. Chuang, U. Kragh-Hansen, and M. Otagiri, “Pharmaceutical strategies utilizing recombinant human serum albumin,” Pharmaceutical Research, vol. 19, no. 5, pp. 569–577, 2002. View at Publisher · View at Google Scholar · View at Scopus
  16. P. A. Dijkmans, R. Senior, H. Becher et al., “Myocardial contrast echocardiography evolving as a clinically feasible technique for accurate, rapid, and safe assessment of myocardial perfusion. The evidence so far,” Journal of the American College of Cardiology, vol. 48, no. 11, pp. 2168–2177, 2006. View at Publisher · View at Google Scholar · View at Scopus
  17. G. Korpanty, S. Chen, R. V. Shohet et al., “Targeting of VEGF-mediated angiogenesis to rat myocardium using ultrasonic destruction of microbubbles,” Gene Therapy, vol. 12, no. 17, pp. 1305–1312, 2005. View at Publisher · View at Google Scholar · View at Scopus
  18. R. Bekeredjian, R. D. Kroll, E. Fein et al., “Ultrasound targeted microbubble destruction increases capillary permeability in hepatomas,” Ultrasound in Medicine and Biology, vol. 33, no. 10, pp. 1592–1598, 2007. View at Publisher · View at Google Scholar · View at Scopus
  19. K. Hynynen, “Ultrasound for drug and gene delivery to the brain,” Advanced Drug Delivery Reviews, vol. 60, no. 10, pp. 1209–1217, 2008. View at Publisher · View at Google Scholar · View at Scopus
  20. C. R. Mayer and R. Bekeredjian, “Ultrasonic gene and drug delivery to the cardiovascular system,” Advanced Drug Delivery Reviews, vol. 60, no. 10, pp. 1177–1192, 2008. View at Publisher · View at Google Scholar · View at Scopus
  21. D. L. Miller, S. V. Pislaru, and J. F. Greenleaf, “Sonoporation: mechanical DNA delivery by ultrasonic cavitation,” Somatic Cell and Molecular Genetics, vol. 27, no. 1-6, pp. 115–134, 2002. View at Publisher · View at Google Scholar · View at Scopus
  22. P. Marmottant and S. Hilgenfeldt, “Controlled vesicle deformation and lysis by single oscillating bubbles,” Nature, vol. 423, no. 6936, pp. 153–156, 2003. View at Publisher · View at Google Scholar · View at Scopus
  23. E. A. Brujan, “The role of cavitation microjets in the therapeutic applications of ultrasound,” Ultrasound in Medicine and Biology, vol. 30, no. 3, pp. 381–387, 2004. View at Publisher · View at Google Scholar · View at Scopus
  24. J. E. Chômas, P. Dayton, J. Alien, K. Morgan, and K. W. Ferrara, “Mechanisms of contrast agent destruction,” IEEE Transactions on Ultrasonics, Ferroelectrics, and Frequency Control, vol. 48, no. 1, pp. 232–248, 2001. View at Publisher · View at Google Scholar · View at Scopus
  25. J. Wu, “Theoretical study on shear stress generated by microstreaming surrounding contrast agents attached to living cells,” Ultrasound in Medicine and Biology, vol. 28, no. 1, pp. 125–129, 2002. View at Publisher · View at Google Scholar · View at Scopus
  26. S. Chen, R. V. Shohet, R. Bekeredjian, P. Frenkel, and P. A. Grayburn, “Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound-targeted microbubble destruction,” Journal of the American College of Cardiology, vol. 42, no. 2, pp. 301–308, 2003. View at Publisher · View at Google Scholar · View at Scopus