Abstract
BackgroundVascular 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.FindingsRibozymes, 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.ConclusionThese 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.
Highlights
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
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
This paper describes the design and experimentation of a hammerhead ribozyme-mediated gene knock down strategy able to dissect PDGF-BB signaling pathway in order to evidence how a single receptor can activate alternative signaling cascades emerging as important switch in VSMC phenotype modulation
Summary
With the administration of hammerhead ribozymes, a significant reduction of the PDGFR-β target mRNA was obtained. This treatment appeared devoid of any toxic side effects and considerably affected the PDGF-BB signaling activity as revealed by biological assays. Migration activity in ribozyme-treated cultures, monitored by wound assay, fell down to 10% of levels attributed to PDGF stimulated cells. The specificity of such a knocking down effect was confirmed by the very small inhibition induced by similar administration of the inactive ribozyme. A possible interpretation of these data is that the PDGFR-β partial knock down causes an unbalance among the different PDGF receptors. Competing interests The authors declare that they have no competing interests
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