Role of E2F1-Cyclin E1-Cyclin E2 Circuit in Human Coronary Smooth Muscle Cell Proliferation and Therapeutic Potential of Its Downregulation by siRNAs

Abstract

Aberrant coronary vascular smooth muscle cell (CSMC) proliferation is a pivotal event underlying intimal hyperplasia, a phenomenon impairing the long-term efficacy of bypass surgery and angioplasty procedures. Consequently research has become focused on efforts to identify molecules that are able to control CSMC proliferation. We investigated downregulation of CSMC growth by small interfering RNAs (siRNAs) targeted against E2F1, cyclin E1, and cyclin E2 genes, whose contribution to CSMC proliferation is only now being recognized. Chemically synthesized siRNAs were delivered by two different transfection reagents to asynchronous and synchronous growing human CSMCs cultivated either in normo-or hyperglycemic conditions. The depletion of each of the three target genes affected the expression of the other two genes, demonstrating a close regulatory control. The clearest effects associated with the inhibition of the E2F1-cyclin E1/E2 circuit were the reduction in the phosphorylation levels of the retinoblastoma protein pRB and a decrease in the amount of cyclin A2. At the phenotypic level the downmodulation of CSMC proliferation resulted in a decrease of S phase matched by an increase of G1-G0 phase cell amounts. The antiproliferative effect was cell-donor and transfectant independent, reversible, and effective in asynchronous and synchronous growing CSMCs. Importantly, it was also evident in hyperglycemia, a condition that underlies diabetes. No significant aspecific cytotoxicity was observed. Our data demonstrate the interrelation among E2F1-cyclin E1-cyclin E2 and the pivotal role this circuit exerts in CSMC proliferation. Additionally, our work validates the concept of utilizing anti-E2F1-cyclin E1-cyclin E2 siRNAs to develop a potential novel therapy to control intimal hyperplasia.

Improving siRNA Bio-Distribution and Minimizing Side Effects

“…We typically perform the selection by testing different amounts of a given lipid, different ratio lipid/siRNA, different transfection times and different transfection medium [103][104][105]. In parallel to the above mentioned tests, we also evaluate the transfection efficiency and siRNA cellular distribution by using FITC-labelled siRNAs.…”

“…In contrast, a more contained proteomic analysis limited to some proteins interacting with the target protein, are technically easier to perform, still being reasonably informative. For example, while studying the effects of the depletion of the transcription factor E2F1 [104], we evaluated also the effects on the protein levels of some of its transcriptional regulated proteins such as cyclin E1 and E2. A higher, although still limited, amount of protein interactions can be evaluated by a recently developed approach.…”

“…To further distinguish between off-targeting and specific siRNA effects other checks may be performed [4,104,[113][114][115][116]. Finally, it has to be excluded the possibility that the siRNA triggers the unspecific expression of genes involved in the activation of the interferon response [117], thus generating a non specific phenotype.…”

Development of a simple, biocompatible and cost-effective Inulin-Diethylenetriamine based siRNA delivery system

“…The sequences of the siRNAs directed against the transcription factor E2F1 mRNA (siE2F1) and the luciferase mRNA (siGL2, control), were purchased from Eurofins Genomics GmBH, Ebersberg, D. The sequences of these chemically un-modified siRNAs, have been previously reported Dapas et al, 2009). The day before transfection, JHH6 cells were seeded at a density of 3.8 Á 10 3 cells/cm 2 in 6 well microplate in the presence of 3 ml of 10% fetal calf serum-containing medium.…”

“…Compared to the conventional therapeutic drugs, siRNAs represent an emerging paradigm for the treatment of many human diseases such as cancer Resnier et al, 2013), cardiovascular, neurodegenerative, metabolic diseases and viral illnesses (Werth et al, 2010;Dapas et al, 2009;Racchi et al, 2009;Deng et al, 2014). However, owing to their negative charges, siRNA molecules are not readily taken up by cells that have a negatively charged surface.…”