7-25-12 UBRP Meeting Presentation

7/27/2019 7-25-12 UBRP Meeting Presentation

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SamirMohandes

July 25, 2012

ENDOTHELIALIZING

VASCULAR GRAFTSWITH PROTEIN

NANOARRAYS


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Number one cause of death amongAmericans

Atherosclerotic coronary heartdisease:

Plaque buildup observed along theinside walls of blood vessels

Low density lipoproteins

Cholesterol

Triglycerides

Coronary arteries become hard andnarrow

Plaque traps platelets, resulting infurther narrowing of the coronaryartery

Blood clots form, cutting off bloodsupply to cardiac muscles

Complete occlusion of the arteryresults in a heart attack

CARDIOVASCULAR DISEASES

Heart disease33%

Cancer31%

Chronic lowerrespiratorydiseases

7%Stroke7%Accidents

6%Alzheimer's

disease4%

Diabetes4%

Influenza andPneumonia

3%

Nephritis,nephrotic

syndrome, andnephrosis

3%

Suicide2%

Leading causes of death in the United States, 2009(Source: Center for Disease Control and Prevention Preliminary Data for 2010)


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PERCUTANEOUS CATHETER INTERVENTION

Percutaneous catheter intervention (PCI) is the most common form of revascularization therapy

Opensblockedarteries

Balloonangioplasty

Long-termstructuralsupport

Drug elutingstent (DES)

deployed

DES remains,releasing drugs

to inhibit cellproliferation

Balloonremoved

Serious risks and limitationscompromise the efficacy of PCI

Stent thrombosis: formation ofblood clots within the stent

Restenosis: re-narrowing of thecoronary artery after treatment

Central to these limitations areplatelet deposition and cellproliferation at the stent site


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UNDERSTANDING THE PROBLEM

Damage to the arterial wallby PCI procedure De-endothelialization Exposure

Thrombogenicity of thestent DES cannot endothelialize

while releasing cytotoxicdrugs

The result: no endothelialcoverage on pro-thromboticzones of the arterial wall

Problems

Anti-platelet agents(clopidogrel)

Solutions

Periodic, unpredictable stentthrombosis

Difficulties with proceduresperformed after DES PCI Cessation of anti-platelet

therapy required Increased risk of stent

thrombosis Many patients are

clopidogrel non -responders

Problems

It is the goal of this project to develop novel methodsthat will address and resolve these limitations


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Endothelial and smooth muscle cells are key whenexamining the de-endothelialization and exposureaspects of revascularization therapy

Smooth muscle cells exist in two phenotypesSynthetic (proliferative)Contractile (functional)

Phenotypic modulation can occur in smooth musclecells, and is influenced by several factors:

GeneticsEnvironmental cues

Biochemical factors

Extracellular matrix componentsPhysical factors

Smooth muscle cells revert to the synthetic phenotypein culture

Application of synthetic smooth muscle cells to

cardiovascular stents increases the risk of restenosis

DEVELOPING NEW SOLUTIONS

Image source: S.S.M. Rensen, P.A.F.M. Doevendans, G.J.J.M. van Eys.Regulation and characteristics of vascular smooth muscle cell phenotypicdiversity. Netherlands Heart Journal 2007;15(3):100-108.


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3T3 mouse fibroblasts were used in theexperiment

Fibroblasts are a type of connective tissue thatsynthesize extracellular matrix and collagen in the

body

Low maintenance cell culture

Reproduce rapidly

Contact guidance: cellular response to underlyingsubstratum topological features on a substrate

Contact guidance can be used to emulate andregulate the spatial cues and cellular signals thatexists in vivo , influencing cell behavior

FIBROBLASTS AND CONTACT GUIDANCE


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MICROPATTERN FABRICATION

1) Spin coat PMMA polymer ontopositively charged glass substrate

2) Electron beam lithography

3) Develop PMMA


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THE MICROPATTERNS: A DETAILED LOOK

Wave 1 Wave 2 Wave 3

10m Lines Pattern Wavelength Amplitude

Wave 1 40m 10m

Wave 2 30m 5m

Wave 3 30m 10m

20m Lines


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Fibroblasts were seeded onto patternedchips and then incubated in 95% air, 5% CO 2at 37C for 4, 24, or 48 hours

TRITC-conjugated Phalloidin dye (red) was

used to image actin filaments

DAPI dye (blue) was used to stain cell nuclei

Imaging performed using an inverted epi-fluorescence microscope at 10x and 60xmagnification

The following measurements were taken:Cell count

Cell length

Cell width

Cell orientation (relative to pattern)

EXPERIMENTAL PROTOCOL


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RESULTS: CELL ALIGNMENT TO PATTERNS


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0%

5%

10%

15%

20%

25%

30%

- 9 0

- 8 5

- 8 0

- 7 5

- 7 0

- 6 5

- 6 0

- 5 5

- 5 0

- 4 5

- 4 0

- 3 5

- 3 0

- 2 5

- 2 0

- 1 5

- 1 0 - 5 0 5 1

0 1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

M o r e

F r e q u e

n c y

Angle (Degrees)

10 um spaced lines


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0%

5%

10%

15%

20%

25%

30%

- 9 0

- 8 5

- 8 0

- 7 5

- 7 0

- 6 5

- 6 0

- 5 5

- 5 0

- 4 5

- 4 0

- 3 5

- 3 0

- 2 5

- 2 0

- 1 5

- 1 0 - 5 0 5 1

0 1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

M o r e

F r e q u e

n c y

Angle (Degrees)

Waves 1


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0%

5%

10%

15%

20%

25%

30%

- 9 0

- 8 5

- 8 0

- 7 5

- 7 0

- 6 5

- 6 0

- 5 5

- 5 0

- 4 5

- 4 0

- 3 5

- 3 0

- 2 5

- 2 0

- 1 5

- 1 0 - 5 0 5 1

0 1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

M o r e

F r e q u e

n c y

Angle (Degrees)

Waves 2


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0%

5%

10%

15%

20%

25%

30%

- 9 0

- 8 5

- 8 0

- 7 5

- 7 0

- 6 5

- 6 0

- 5 5

- 5 0

- 4 5

- 4 0

- 3 5

- 3 0

- 2 5

- 2 0

- 1 5

- 1 0 - 5 0 5 1

0 1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

M o r e

F r e q u e

n c y

Angle (Degrees)

20 um spaced lines


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0%

5%

10%

15%

20%

25%

30%

- 9 0

- 8 5

- 8 0

- 7 5

- 7 0

- 6 5

- 6 0

- 5 5

- 5 0

- 4 5

- 4 0

- 3 5

- 3 0

- 2 5

- 2 0

- 1 5

- 1 0 - 5 0 5 1

0 1 5

2 0

2 5

3 0

3 5

4 0

4 5

5 0

5 5

6 0

6 5

7 0

7 5

8 0

8 5

9 0

M o r e

F r e q u e

n c y

Angle (Degrees)

No Pattern


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Bottom Top


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RESULTS: CHANGES IN CELL DIMENSIONS

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

No Pattern 0% 10um spacing23.11%

20 um spacing18.08%:

Waves 1 40-1024.3%:

Waves 2 30-523.5%:

Waves 3 30-1027.15%:

C e l l L e n g t h ( u m )

Cell Length

24hrs

48hrs


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RESULTS: CHANGES IN CELL DIMENSIONS

0.00

5.00

10.00

15.00

20.00

25.00

No Pattern 0% 10um spacing23.11%

20 um spacing18.08%:

Waves 1 40-1024.3%:

Waves 2 30-523.5%:

Waves 3 30-1027.15%:

C e l l W i d t h ( u m )

Cell Width

24hrs

48hrs


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THE NEXT STEP

Image source: S.S.M. Rensen, P.A.F.M. Doevendans, G.J.J.M. van Eys.Regulation and characteristics of vascular smooth muscle cell phenotypicdiversity. Netherlands Heart Journal 2007;15(3):100-108.


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THE NEXT STEP

Image source: S.S.M. Rensen, P.A.F.M. Doevendans, G.J.J.M. van Eys. Regulation and characteristics of vascular smooth muscle cell phenotypic diversity. Netherlands Heart Journal 2007;15(3):100-108.

Documents

7-25-12 UBRP Meeting Presentation