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

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.

Inhibitory effect of Puerariae radix flavones on platelet-derived growth factor-BB-induced proliferation of vascular smooth muscle cells via PI3K and ERK pathways

“…VSMCs have been shown to switch from a differentiated to a proliferative phenotype upon PDGF-BB stimulation, resulting in the downregulation of the VSMC differentiation markers, α-SMA, smoothelin and desmin ( 9 ). PRF was found to inhibit the PDGF-BB-stimulated proliferation of VSMCs; therefore, the expression levels of the aforementioned markers in the VSMC differentiated phenotype were investigated for each group.…”

“…Under physiological conditions, VSMCs remain in a quiescent state and express α-smooth muscle actin (α-SMA), desmin and smoothelin ( 8 ). However, in response to various stimuli, such as PDGF-BB, VSMCs may switch to a highly proliferative state, resulting in decreased expression levels of these markers ( 9 ). Furthermore, the cell cycle progression and expression levels of cell cycle-associated proteins have been found to be upregulated by PDGF-BB in VSMCs ( 10 ).…”

Vascular Smooth Muscle Cells activation revealed by quantitative phosphoproteomics analysis

“…Following growth factor binding to its receptor, a cascade of kinases and phosphatases are activated and, within a few minutes, a series of events and speci c responses are triggered while the phosphorylation state of the proteins changes dramatically [9]. ese early events, even if phenotypically imperceptible, are decisive for the activation process in VSMCs [6,10], and result with a complete reorganization of the cell structure.…”