Abnormal proliferation of vascular smooth muscle cells (VSMCs) contributes to intimal hyperplasia during atherosclerosis and restenosis, but the endogenous cell cycle regulatory factors underlying VSMC growth in response to arterial injury are not well understood. In the present study, we report that downregulation of cyclin-dependent kinase 2 (cdk2) activity in serum-deprived VSMCs was associated with the formation of complexes between cdk2 and its inhib-

Excessive proliferation of vascular smooth muscle cells (VSMCs) contributes to vessel renarrowing after angioplasty. Here we investigated the transcriptional regulation of the cyclin A gene, a key positive regulator of S phase that is induced after angioplasty. We show that Ras-dependent mitogenic signaling is essential for the normal stimulation of cyclin A promoter activity and DNA synthesis in VSMCs. Overexpression of the AP-1 transcription factor c-fos can circumvent this requirement via interaction with the cAMP-responsive element (CRE) in the cyclin A promoter. Moreover, c-fos overexpression in serum-starved VSMCs results in the induction of cyclin A promoter activity in a CRE-dependent manner, and increased binding of endogenous c-fos protein to the cyclin A CRE precedes the onset of DNA replication in VSMCs induced by serum in vitro and by angioplasty in vivo. We also show that E2F function is essential for both serum- and c-fos-dependent induction of cyclin A expression. Taken together, these findings suggest that c-fos and E2F are important components of the signaling cascade that link Ras activity to cyclin A transcription in VSMCs. These studies illustrate a novel link between the transcriptional and cell cycle machinery that may be relevant to the pathogenesis of vascular proliferative disorders.

Abdominal aortic aneurysms (AAAs) are rupture-prone progressive dilations of the infrarenal aorta due to a loss of elastic matrix that lead to weakening of the aortic wall. Therapies to coax biomimetic regenerative repair of the elastic matrix by resident, diseased vascular cells may thus be useful to slow, arrest, or regress AAA growth. Hyaluronan oligomers (HA-o) have been shown to induce elastic matrix synthesis by healthy and aneurysmal rat aortic smooth muscle cells (SMCs) in vitro but only via exogenous dosing, which potentially has side effects and limitations to in vivo delivery towards therapy. In this paper, we describe the development of HA-o loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs) for targeted, controlled, and sustained delivery of HA-o towards the elastogenic induction of aneurysmal rat aortic SMCs. These NPs were able to deliver HA-o over an extended period (>30 days) at previously determined elastogenic doses (0.2 – 20 μg/mL). HA-o released from the NPs led to dose dependent increases in elastic matrix synthesis, and the recruitment and activity of lysyl oxidase (LOX), the enzyme which crosslinks elastin precursor molecules into mature fibers/matrix. Therefore, we were able to successfully develop a nanoparticle based system for controlled and sustained HA-o delivery for the in vitro elastogenic induction of aneurysmal rat aortic smooth muscle cells (EaRASMCs).

Abdominal aortic aneurysms (AAAs) involve chronic overexpression of proteases in the aortic wall that result in disruption of elastic fibers and consequent loss of vessel elasticity. Nearly 75% of AAAs contain flow-obstructing, fibrin-rich intraluminal thrombi (ILT), which act as a) a bioinert shield, protecting the underlying AAA wall from high hemodynamic stresses, and b) a reservoir of inflammatory cells and proteases that cause matrix breakdown. For these reasons, restoring flow through the aorta lumen and facilitating transmural diffusion of therapeutics from circulation to the AAA wall must be achieved by slow thrombolysis of the ILT to render it porous without rapid breakdown. Intravenously dosed tissue plasminogen activator (tPA) has been shown to rapidly lyse ILTs in acute stroke and myocardial infarctions. For future use in opening up AAA segments, in this study, we investigated the ability of tPA released from poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs) to slowly lyse fibrin clots without inducing proteolytic injury and matrix synthesis-inhibitory effects on cultured rat aneurysmal smooth muscle cells (EaRASMCs). Fibrin clot lysis time was greatly extended over that in presence of exogenous tPA. Surface functionalization of NPs with a cationic amphiphile allowed them to bind to anionic fibrin clot, release tPA at a slower rate and to lyse the clot as a front proceeding outwards in unlike the more rapid and homogenous lysis that occurred due to anionic PLGA NPs. Elastic matrix content was decreased in EaRASMC cultures exposed to byproducts of clot lysis with exogenous tPA, but not tPA-NPs, and was likely due to increased proteolytic activity (MMPs, plasmin) in EaRASMC cultures exposed to exogenous tPA-lysed clots. Our results suggest that gradual ILT lysis via slow release of tPA from NPs will be likely beneficial over exogenous tPA delivery in preserving elastic matrix content and attenuating matrilysis in the adjoining AAA wall, in vivo, while rendering the ILT porous to facilitate transmural delivery of endoluminally delivered AAA therapeutics.