• 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 hyperplasiaaffected by multiple factors

Endothelially derived -vasodilating agents

- relaxation agentsvasoconstrictors

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 cellsEnhance SMC migrationpromote SMC proliferationencourage unhealthy vascular

remodelingData 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 (3.0m

pore) membrane no cell contact

EC

SMCs

Pressure apparatus

Custom-made chamber 130mm Hg monitored

with manometer 95% room air and 5%CO2

370C temperature-humidity

daily media pH measurements

Coculture model

EC

SMCs

Extracellular pressure130-135 mmHg

Four pressure-culture conditions

SMCs

EC

SMCs

EC

SMCs SMCs

130mmHg

130mmHg

Effect of pressure on EC/SMC coculture

C-myc dependentLate entry to S-Phase

Vouyouka et al JSR 2003

SMCs stained with TUNELSMC/0 SMC/0-P

SMC/EC-PSMC/EC

Pressure and coculture enhancesSMC apoptosis

Vouyouka et al Surgery 2004

0.0

5.0

10.0

15.0

20.0

25.0

SMC/0 SMC/EC SMC/0-P SMC/EC-papoptotic index(%)

***

*

Topisomerase IIaFas- Fas Ligand

Effect of pressure and coculture on SMC Topoisomerase II

0.0

0.4

0.8

1.2

1.6

*

SMC/0 SMC/EC SMC/0-P SMC/EC-P

Topo II

Actin

*

Effect of pressure and coculture on SMC Topoisomerase II

0.0

0.4

0.8

1.2

1.6

*

SMC/0 SMC/EC SMC/0-P SMC/EC-P

Topo II

Actin

*

i n O S l e v e l s i n S M C s - w e s t e r n b l o t a n a l y s i s

0

0 . 5

1

1 . 5

2

2 . 5

3

3 . 5

S M C / 0 S M C / E C S M C / 0 - P S M C / E C - P

iNOS/actin ratio

*

Pressure and coculture increases iNOS

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

NP DMSOP DMSO

NP Calphostine P CalphostineNP PD98059P PD98059NP L-NameP L-Namenormalised densitometric ratio eNOS/Actin

**

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

Day 5 (c-myc)n=7

0

0.2

0.4

0.6

0.8

1

1.2

1.4

SMC,NP SMC/EC,NP SMC,P SMC/EC,P

c-mycactin

P<0.03

Vouyouka et al JSR 2003

3D Structured PLA Scaffolds

Structured mechanical compliance3-D architecture3-D architecture Controlled ECM ChemistryControlled ECM Chemistry

Tissue Tissue EngineeringEngineering

Tissue Tissue ReplacementReplacement

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.