Abstract 5815: Therapeutic Targeting of the Calmodulin-Cyclin E Interaction in Vascular Smooth Muscle Cells
Background: Cell cycle regulation in vascular smooth muscle cells (VSMC) is a putative target in proliferative vascular diseases. Having shown that increases in intracellular [Ca2+] are tightly coordinated and critical for VSMC proliferation, we also discovered that binding of Ca2+/calmodulin (CaM) to cyclin E/cdk2 is necessary for Ca2+-sensitive G1-to-S phase progression in VSMC. To test the importance of this, we developed a synthetic CaM-binding sequence (CBS) that blocked CaM-cyclin E interactions, prevented activation of cdk2, and abrogated Ca2+-sensitive G1-to-S transitions in VSMC. Of note, binding of CaM to another target protein, calcineurin, was not altered by CBS.
Objective: To evaluate the physiological relevance of targeting CaM-cyclin E interactions in human VSMC in vitro and in a mouse model of neointima formation in vivo.
Methods & Results: Human aortic SMC (HA-SMC) were treated with the CBS peptide conjugated to the viral TAT transduction domain to increase bioavailability. Peptide delivery of TAT-CBS was confirmed by immunochemistry. TAT-CBS caused a dose-dependent anti-proliferative effect in HA-SMC, as well as in human aortic endothelial cells and mouse embryonic fibroblasts (MEF) as measured by 3H-thymidine incorporation. Importantly, these effects were lost in MEF from cyclin E1/E2 double knockout mice, further supporting target specificity for CaM/cyclin E. Moreover, TAT-CBS treatment was not cytotoxic, as it did not increase either LDH or caspase-3 release compared to controls. In vivo application of TAT-CBS in pluronic gel to the outside of injured mouse carotid arteries significantly decreased neointima formation and medial thickening without affecting re-endothelialization as compared to animals treated with vehicle-only or negative-control peptide.
Summary: Inhibition of CaM binding to cyclin E/cdk2 with the TAT-CBS peptide significantly decreases HA-SMC proliferation in a dose-dependent manner in vitro. Moreover, application of TAT-CBS-containing gel to injured carotid arteries inhibits VSMC proliferation without affecting re-endothelialization. These data support
TAT-CBS as a novel therapy for restenosis, and
the general approach of targeting Ca2+-sensitive cell cycle control in VSMC.