Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells

BMC Research Notes

Antonella Cecchettini⁴  Maria Giovanna Trivella¹  Lorena Tedeschi¹  Silvia Rocchiccioli¹  Milena Rizzo¹  Alberto Mercatanti¹  Lorenzo Citti¹  Claudia Boccardi²  Caterina Lande³ 

[1] Institute of Clinical Physiology, CNR, Pisa, Italy;Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy;Istitute of Clinical Physiology, National Research Council – IFC-CNR, Via G. Moruzzi, 1, Pisa, 56124, Italy;Department of Human Morphology and Applied Biology, University of Pisa, Pisa, Italy

关键词: Functional proteomics;    Cardiovascular disease;    Vascular smooth muscle cells;    Hammerhead ribozyme;   

Others  :  1166370
DOI  :  10.1186/1756-0500-5-268

 received in 2012-02-19, accepted in 2012-06-07,  发布年份 2012

【 摘 要 】

Background

Vascular Smooth Muscle Cells (VSMCs), due to their plasticity and ability to shift from a physiological contractile-quiescent phenotype to a pathological proliferating-activated status, play a central role in the onset and progression of atherosclerosis and cardiovascular diseases. PDGF-BB, among a series of cytokines and growth factors, has been identified as the critical factor in this phenotypic switch. In order to obtain new insights on the molecular effects triggered by PDGF-BB, a hammerhead ribozyme targeting the membrane receptor PDGFR-β was applied to inhibit PDGF pathway in porcine VSMCs.

Findings

Ribozymes, loaded on a cationic polymer-based vehicle, were delivered into cultured VSMCs. A significant impairment of the activation mechanisms triggered by PDGF-BB was demonstrated since cell migration decreased after treatments. In order to functionally validate the effects of PDGFR-β partial knock down we focused on the phosphorylation status of two proteins, protein disulfide isomerase-A3 (PDI-A3) and heat shock protein-60 (HSP-60), previously identified as indicative of VSMC phenotypic switch after PDGF-BB stimulation. Interestingly, while PDI-A3 phosphorylation was counteracted by the ribozyme administration indicating that PDI-A3 is a factor downstream the receptor signalling cascade, the HSP-60 phosphorylation status was greatly increased by the ribozyme administration.

Conclusion

These contradictory observations suggested that PDGF-BB might trigger different parallel pathways that could be modulated by alternative isoforms of the receptors for the growth factor. In conclusion the knock down strategy here described enables to discriminate between two tightly intermingled pathways. Moreover it opens new attractive perspectives in functional investigations where combined gene knock down and proteomic technologies would allow the identification of key factors and pathways involved in VSMC-linked pathological disorders.

【 授权许可】

   
2012 Lande et al.; licensee BioMed Central Ltd.

【 图 表 】

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【 参考文献 】

• [1]Owens GK, Kumar MS, Wamhoff BR: Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004, 84:767-801.
• [2]Gerthoffer WT: Mechanisms of vascular smooth muscle cell migration. Circ Res 2007, 100:607-621.
• [3]Davies MG, Owens EL, Mason DP, Lea H, Tran PK, Vergel S, Hawkins SA, Hart CE, Clowes AW: Effect of platelet-derived growth factor receptor-alpha and -beta blockade on flow-induced neointimal formation in endothelialized baboon vascular grafts. Circ Res 2000, 86:779-786.
• [4]Tanizawa S, Ueda M, vanderLoos CM, vanderWal AC, Becker AE: Expression of platelet derived growth factor B chain and beta receptor in human coronary arteries after percutaneous transluminal coronary angioplasty: An immunohistochemical study. Heart 1996, 75:549-556.
• [5]Cecchettini A, Rocchiccioli S, Boccardi C, Citti L: Vascular smooth muscle-cell activation: proteomics point of view. Int Rev Cell Mol Biol 2011, 288:43-99.
• [6]Lowe HC, Schwartz RS, Mac Neill BD, Jang LK, Hayase M, Rogers C, Oesterle SN: The porcine coronary model of in-stent restenosis: current status in the era of drug-eluting stents. Catheter Cardiovasc Interv 2003, 60:515-523.
• [7]Bhindi R, Fahmy RG, Lowe HC: Brothers in arms - DNA enzymes, short interfering RNA, and the emerging wave of small-molecule nucleic acid-based gene-silencing strategies. American J Pathology 2007, 171:1079-1088.
• [8]Tedeschi L, Lande C, Cecchettini A, Citti L: Hammerhead ribozymes in therapeutic target discovery and validation. Drug Discov Today 2009, 14:776-783.
• [9]Phylactou LA, Tsipouras P, Kilpatrick MW: Hammerhead ribozymes targeted to the FBN1 mRNA can discriminate a single base mismatch between ribozyme and target. Biochem Biophys Res Commun 1998, 249:804-810.
• [10]Citti L, Rainaldi G: Synthetic hammerhead ribozymes as therapeutic tools to control disease genes. Curr Gene Ther 2005, 5:11-24.
• [11]Mercatanti A, Rainaldi G, Mariani L, Marangoni R, Citti L: A method for prediction of accessible sites on an mRNA sequence for target selection of hammerhead ribozymes. J Comput Biol 2002, 9:641-653.
• [12]Citti L, Rovero P, Colombo MG, Mariani L, Poliseno L, Rainaldi G: Efficacy of an amphipathic oligopeptide to shuttle and release a cis-acting DNA decoy into human cells. Biotechniques 2002, 32:172-+.
• [13]Christen T, Bochaton-Piallat ML, Neuville P, Rensen S, Redard M, van Eys G, Gabbiani G: Cultured porcine coronary artery smooth muscle cells - a new model with advanced differentiation. Circ Res 1999, 85:99-107.
• [14]Rocchiccioli S, Citti L, Boccardi C, Ucciferri C, Tedeschi L, Lande C, Trivella MG, Cecchettini A: A gel-free approach in vascular smooth muscle cell proteome: perpectives for a better insight into activation. Proteome Science 2010, 8:1-15. BioMed Central Full Text
• [15]Görg A, Obermaier C, Boguth G, Weiss W: Recent developments in two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up to pH 12, longer separation distances and simplified procedures. Electrophoresis 1999, 20:712-717.
• [16]Hochstrasser DF, Patchornik A, Merril CR: Development of polyacrylamide gels that improve the separation of proteins and their detection by silverstaining. Anal Biochem 1988, 173:412-423.
• [17]Blalock TD, Yuan R, Lewin AS, Schultz GS: Hammerhead ribozyme targeting connective tissue growth factor mRNA blocks transforming growth factor-beta mediated cell proliferation. Exp Eye Res 2004, 2004(78):1127-1136.
• [18]Citti L, Boldrini L, Nevischi S, Mariani L, Rainaldi G: Quantitation of in vitro activity of synthetic trans-acting ribozymes using HPLC. Biotechniques 1997, 23:898-903.
• [19]Chilakamarthi U, Murherjee SK, Deb JK: Intervention of geminiviral replication in yeast by ribozyme mediated downregulation of its Rep protein. FEBS Lett 2007, 581:2675-2683.
• [20]Eliyahu H, Barenholz Y, Domb AJ: Polymers for DNA delivery. Molecules 2005, 10:34-64.
• [21]Prochiantz A: Messenger proteins: homeoproteins, TAT and others. Curr Opin Cell Biol 2000, 12:400-406.
• [22]Morris MC, Deshayes S, Heitz F, Divita G: Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol Cell 2008, 100:201-217.
• [23]Giannini CD, Roth WK, Piiper A, Zeuzem S: Enzymatic and antisense effects of a specific anti-Ki-ras ribozyme in vitro and in cell culture. Nucleic Acids Res 1999, 27:2737-2744.
• [24]Boccardi C, Cecchettini A, Caselli A, Camici G, Evangelista M, Mercatanti A, Rainaldi G, Citti L: A proteomic approach to the investigation of early events involved in the activation of vascular smooth muscle cells. Cell Tissue Res 2007, 329:119-128.
• [25]Snoeckx L, Cornelussen RN, Van Nieuwenhoven FA, Reneman RS, Van Der Vusse GJ: Heat shock proteins and cardiovascular pathophysiology. Physiol Rev 2001, 81:1461-1497.
• [26]Pockley AG, de Faire U, Kiessling R, Lemne C, Thulin T, Frostegard J: Circulating heat shock protein and heat shock protein antibody levels in established hypertension. J Hypertens 2002, 20:1815-1820.
• [27]Barazi HO, Zhou LG, Templeton NS, Krutzsch HC, Roberts DD: Identification of heat shock protein 60 as a molecular mediator of alpha 3 beta 1 integrin activation. Cancer Res 2002, 62:1541-1548.
• [28]Kelly L, Asquith RM, Baleato EA, McLaughlin BN, Aitken NJ: Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition. J Cell Sci 2004, 117:3645-3657.
• [29]Turano C, Coppari S, Altieri F, Ferraro A: Proteins of the PDI family: unpredicted non-ER locations and functions. J Cell Physiol 2002, 193:154-163.
• [30]Goplen D, Wang J, Enger PØ, Tysnes BB, Terzis AJ, Laerum OD, Bjerkvig R: Protein disulfide isomerase expression is related to the invasive properties of malignant glioma. Cancer Res 2006, 66:9895-9902.
• [31]Lahav J, Wijnen EM, Hess O, Hamaia SW, Griffiths D, Makris M, Knight CJ, Essex DW, Farndale RW: Enzymatically catalyzed disulfide exchange is required for platelet adhesion to collagen via integrin α2β1. Blood 2003, 102:2085-2092.
• [32]Lahav J, Gofer-Dadosh N, Luboshitz J, Hess O, Shaklai M: Protein disulfide isomerase mediates integrin dependent adhesion. FEBS Lett 2000, 475:89-92.
• [33]Hogg PJ, Hotchkiss KA, Jimenez BM, Stathakis P, Chesterman CN: Interaction of platelet-derived growth factor with thrombospondin 1. Biochem J 1997, 326:709-716.
• [34]Yu J, Deuel TF, Kim H-RC: Platelet-derived Growth Factor (PDGF) Receptor-α Activates c-Jun NH2-terminal Kinase-1 and Antagonizes PDGF Receptor-β-induced Phenotypic Transformation. J Biol Chem 2000, 275:19076-19082.