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

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-...

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Autores principales: Lande, Caterina, Boccardi, Claudia, Citti, Lorenzo, Mercatanti, Alberto, Rizzo, Milena, Rocchiccioli, Silvia, Tedeschi, Lorena, Trivella, Maria Giovanna, Cecchettini, Antonella
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2012
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Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393606/
https://www.ncbi.nlm.nih.gov/pubmed/22676333
http://dx.doi.org/10.1186/1756-0500-5-268
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author Lande, Caterina
Boccardi, Claudia
Citti, Lorenzo
Mercatanti, Alberto
Rizzo, Milena
Rocchiccioli, Silvia
Tedeschi, Lorena
Trivella, Maria Giovanna
Cecchettini, Antonella
author_facet Lande, Caterina
Boccardi, Claudia
Citti, Lorenzo
Mercatanti, Alberto
Rizzo, Milena
Rocchiccioli, Silvia
Tedeschi, Lorena
Trivella, Maria Giovanna
Cecchettini, Antonella
author_sort Lande, Caterina
collection PubMed
description 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.
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spelling pubmed-33936062012-07-11 Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells Lande, Caterina Boccardi, Claudia Citti, Lorenzo Mercatanti, Alberto Rizzo, Milena Rocchiccioli, Silvia Tedeschi, Lorena Trivella, Maria Giovanna Cecchettini, Antonella BMC Res Notes Short Report 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. BioMed Central 2012-07-10 /pmc/articles/PMC3393606/ /pubmed/22676333 http://dx.doi.org/10.1186/1756-0500-5-268 Text en Copyright ©2012 Lande et al.; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Short Report
Lande, Caterina
Boccardi, Claudia
Citti, Lorenzo
Mercatanti, Alberto
Rizzo, Milena
Rocchiccioli, Silvia
Tedeschi, Lorena
Trivella, Maria Giovanna
Cecchettini, Antonella
Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells
title Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells
title_full Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells
title_fullStr Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells
title_full_unstemmed Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells
title_short Ribozyme-mediated gene knock down strategy to dissect the consequences of PDGF stimulation in vascular smooth muscle cells
title_sort ribozyme-mediated gene knock down strategy to dissect the consequences of pdgf stimulation in vascular smooth muscle cells
topic Short Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3393606/
https://www.ncbi.nlm.nih.gov/pubmed/22676333
http://dx.doi.org/10.1186/1756-0500-5-268
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