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-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.


RESULTS
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.


CONCLUSIONS
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.
Institute of Clinical Physiology, CNR, Pisa, Italy. caterina.lande@yahoo.it
Caterina
C
Lande
Claudia
C
Boccardi
Lorenzo
L
Citti
Alberto
A
Mercatanti
Milena
M
Rizzo
Silvia
S
Rocchiccioli
Lorena
L
Tedeschi
Maria Giovanna
MG
Trivella
Antonella
A
Cecchettini
D000818:Animals; D000077214:Becaplermin; D015153:Blotting, Western; D002465:Cell Movement / Q000235:genetics / Q000502:physiology; D002478:Cells, Cultured; D018834:Chaperonin 60 / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D015870:Gene Expression; D055785:Gene Knockdown Techniques / Q000379:methods*; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D010766:Phosphorylation; D019704:Protein Disulfide-Isomerases / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D020574:Proto-Oncogene Proteins c-sis / Q000378:metabolism / Q000502:physiology*; D016337:RNA, Catalytic / Q000235:genetics*; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics / Q000502:physiology; D034421:Sus scrofa; D014162:Transfection
D018834:Chaperonin 60; D020574:Proto-Oncogene Proteins c-sis; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D000077214:Becaplermin; D020797:Receptor, Platelet-Derived Growth Factor beta; D019704:Protein Disulfide-Isomerases