excessive SMC growth within the vascular wall after trauma -proliferation -apoptosis -migration - accelerated matrix deposition major cause of failure of endovascular and vascular reconstructions so far very difficult to treat Intimal hyperplasia

Trauma Platelet aggregation Hemodynamic factors Humoral factors altered SMC-EC interaction Intimal hyperplasia affected by multiple factors

Endothelially derived -vasodilating agents - relaxation agents vasoconstrictors Cell to cell interaction? Intimal hyperplasia altered SMC-EC interaction

Intimal hyperplasia in stent -restenosis-

Intimal hyperplasia -restenosis-

Intimal hyperplasia -restenosis management challenging

intimal hyperplasia and EC / SMC interaction are difficult to study in in vivo studies we cannot separate hemodynamic, intercellular and humoral effects In in vitro studies cannot reproduce clinical effects of EC/SMC interaction

EC / SMC interaction is difficult to study In in vivo stable conditions intact confluent ECs control intimal hyperplasia by: -enhanced production of NO -decreased production of endothelin - production of anti inflammatory agents such as heparan sulfate regenerating ECs may have an opposite effect

in vitro conventional cocultures Endothelial cells Enhance SMC migration promote SMC proliferation encourage unhealthy vascular remodeling Data from conventional cocultures do not allow direct translation of the results Powell, Cronewett, et al

There is need for in vitro vasculature models Hemodynamic factors: shear stress, pressure Matrix Physiologic features of the vascular wall -compliance, elasticity

Coculture model Rat aortic SMCs in wells Rat aortic ECs in inserts inserts transferred into wells at 24 hrs semipermeable (3.0  m pore) membrane no cell contact EC SMCs

Pressure apparatus Custom-made chamber 130mm Hg monitored with manometer 95% room air and 5%CO C temperature- humidity daily media pH measurements

Coculture model EC SMCs Extracellular pressure mmHg

Four pressure-culture conditions SMCs EC SMCs EC SMCs 130mmHg

Effect of pressure on EC/SMC coculture C-myc dependent Late entry to S-Phase Vouyouka et al JSR 2003

SMCs stained with TUNEL SMC/0 SMC/0-P SMC/EC-P SMC/EC

Pressure and coculture enhances SMC apoptosis Vouyouka et al Surgery 2004 Topisomerase IIa Fas- Fas Ligand

Effect of pressure and coculture on SMC Topoisomerase II  * SMC/0 SMC/EC SMC/0-P SMC/EC-P Topo II  Actin *

Effect of pressure and coculture on SMC Topoisomerase II  * SMC/0 SMC/EC SMC/0-P SMC/EC-P Topo II  Actin *

* Pressure and coculture increases iNOS

Extracellular pressure is a major regulator of EC/SMC interaction Regulates cocultured SMC growth by enhancing apoptosis and inhibiting SMC proliferation. Mediates these effects mostly through endothelial paracrine function that involves NO and endothelin mediation

Electrospinning fibers with different chemistry and collecting as a single fabric on a rotating mandrel What about vascular wall compliance?

Project Aims Synthesize and characterize bioactive PLA scaffolds containing graded layers of variable mechanical compliance, mimicking the compliance of normal and diseased vessels. (Frey) Investigate endothelial cell and vascular smooth muscle cell adhesion, migration, and proliferation within the stratified layers of the matrices in Aim 1, relating the cell behavior to matrix mechanics. (Reinhart-King) Adapt the scaffold in Aim 1 to support an endothelial and vascular smooth muscle cell co-culture in different pressure environments to investigate the effect of endothelial cells on vascular smooth muscle cell behavior. (Vouyouka and Reinhart-King)

Future aims Incorporate to the in vitro vasculature model stents? try to “treat” the “pathologic” in vitro model with the incorporation -NO bubbles or donors - endothelin receptor antagonists - Hyper cooling of the model - ?????

C-myc levels in SMC cultures c-myc actin P<0.03 Vouyouka et al JSR 2003

3D Structured PLA Scaffolds Structured mechanical compliance 3-D architecture Controlled ECM Chemistry Tissue Engineering Tissue Replacement Disease Models for Prevention Schematic of our approach to integrate princples of tissue engineering to create a scaffold for models of atherosclerosis and disease. Shaded areas identify novel areas addressed in our project.