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.
Highlights
In adult blood vessels, vascular smooth muscle cells are mainly involved in controlling vessel tone to regulate blood flow distribution and blood pressure
Uptake studies indicated that both transfection reagents considered (Cellfectin and Metafectene) had an excellent transfection efficacy in coronary smooth muscle cell (CSMC) as evaluated by using a 5′fluorescein (FITC)-labeled siRNAGL2
Comparable results were obtained using the most active small interfering RNAs (siRNAs), in the presence of the transfection reagent Metafectene (Figure 1B)
Summary
Vascular smooth muscle cells are mainly involved in controlling vessel tone to regulate blood flow distribution and blood pressure. Whereas augmented growth rate is a relevant physiological process in vascular repair, its aberrant increase has been implicated in the pathogenesis of various hyperproliferative vascular diseases such as vein graft occlusion, coronary bypass surgery, in-stent restenosis (ISR), atherosclerosis, and hypertension [2] In these conditions, vascular smooth muscle cell proliferation, together with a number of other pathophysiological events [3], is responsible for the thickening of the intimal vessel layer (intimal hyperplasia [IH]) with the consequent reduction of blood flow. In the case of coronary ISR [3], the downregulation of coronary smooth muscle cell (CSMC) growth by potent antiproliferative and proapoptotic drugs released from endoluminal stents (drug-eluting stent) has been shown to significantly reduce IH [4] This benefit is limited to low-risk patients, siRNA INHIBITION OF CSMC PROLIFERATION with discrete, de novo lesions in native coronary vessels. The understanding of the molecular mechanisms controlling vascular smooth muscle cell proliferation, a pivotal event in IH generation, is crucial in the management of ISR as well as in other hyperproliferative vascular diseases
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