excessive smc growth within the vascular wall after trauma -proliferation -apoptosis -migration -...
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• 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
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Trauma Platelet aggregation Hemodynamic factors Humoral factors altered SMC-EC interaction
Intimal hyperplasiaaffected by multiple factors
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Endothelially derived -vasodilating agents
- relaxation agentsvasoconstrictors
Cell to cell interaction?
Intimal hyperplasia altered SMC-EC interaction
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Intimal hyperplasia in stent -restenosis-
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Intimal hyperplasia
-restenosis-
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Intimal hyperplasia -restenosis
management challenging
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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
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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
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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
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There is need for in vitro vasculature models
Hemodynamic factors: shear stress, pressure
Matrix Physiologic features of the vascular
wall -compliance, elasticity
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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
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Pressure apparatus
Custom-made chamber 130mm Hg monitored
with manometer 95% room air and 5%CO2
370C temperature-humidity
daily media pH measurements
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Coculture model
EC
SMCs
Extracellular pressure130-135 mmHg
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Four pressure-culture conditions
SMCs
EC
SMCs
EC
SMCs SMCs
130mmHg
130mmHg
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Effect of pressure on EC/SMC coculture
C-myc dependentLate entry to S-Phase
Vouyouka et al JSR 2003
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SMCs stained with TUNELSMC/0 SMC/0-P
SMC/EC-PSMC/EC
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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
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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
*
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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
*
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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
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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
**
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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
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Electrospinning fibers with different chemistry and collecting as a single fabric on a rotating mandrel
What about vascular wall compliance?
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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)
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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- ?????
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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
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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.