ENDOTHELIALIZING VASCULAR GRAFTS WITH PROTEIN NANOARRAYS

Samir Mohandes July 25, 2012

CARDIOVASCULAR DISEASES
Number one cause of death among Americans Atherosclerotic coronary heart disease:
Plaque buildup observed along the inside walls of blood vessels
Low density lipoproteins Cholesterol Triglycerides
Heart disease 33% Cancer 31%

Leading causes of death in the United States, 2009

(Source: Center for Disease Control and Prevention Preliminary Data for 2010)

Coronary arteries become hard and narrow Plaque traps platelets, resulting in further narrowing of the coronary artery Blood clots form, cutting off blood supply to cardiac muscles Complete occlusion of the artery results in a heart attack

Suicide 2% Nephritis, nephrotic syndrome, and nephrosis 3% Influenza and Pneumonia 3% Diabetes Alzheimer's 4% disease 4% Accidents 6%

Chronic lower respiratory diseases Stroke 7% 7%

PERCUTANEOUS CATHETER INTERVENTION

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

Balloon angioplasty

Opens blocked arteries

Drug eluting stent (DES) deployed

Long-term structural support

Balloon removed

DES remains, releasing drugs to inhibit cell proliferation

Serious risks and limitations compromise the efficacy of PCI

Stent thrombosis: formation of blood clots within the stent Restenosis: re-narrowing of the coronary artery after treatment

Central to these limitations are platelet deposition and cell proliferation at the stent site

UNDERSTANDING THE PROBLEM

Damage to the arterial wall by PCI procedure De-endothelialization Exposure Thrombogenicity of the stent DES cannot endothelialize while releasing cytotoxic drugs The result: no endothelial coverage on pro-thrombotic zones of the arterial wall

Solutions
Anti-platelet agents (clopidogrel) Periodic, unpredictable stent thrombosis Difficulties with procedures performed after DES PCI Cessation of anti-platelet therapy required Increased risk of stent thrombosis Many patients are clopidogrel non-responders

Problems

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

DEVELOPING NEW SOLUTIONS

Endothelial and smooth muscle cells are key when examining the de-endothelialization and exposure aspects of revascularization therapy Smooth muscle cells exist in two phenotypes
Synthetic (proliferative) Contractile (functional)

Phenotypic modulation can occur in smooth muscle cells, and is influenced by several factors:
Genetics Environmental cues
Biochemical factors Extracellular matrix components Physical factors

Smooth muscle cells revert to the synthetic phenotype in culture Application of synthetic smooth muscle cells to cardiovascular stents increases the risk of restenosis

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.

FIBROBLASTS AND CONTACT GUIDANCE

3T3 mouse fibroblasts were used in the experiment Fibroblasts are a type of connective tissue that synthesize extracellular matrix and collagen in the body Low maintenance cell culture Reproduce rapidly Contact guidance: cellular response to underlying substratum topological features on a substrate Contact guidance can be used to emulate and regulate the spatial cues and cellular signals that exists in vivo, influencing cell behavior

MICROPATTERN FABRICATION
1) Spin coat PMMA polymer onto positively charged glass substrate

2) Electron beam lithography

3) Develop PMMA

THE MICROPATTERNS: A DETAILED LOOK

Wave 1 Wave 2 Wave 3

10m Lines

20m Lines

Pattern Wave 1 Wave 2 Wave 3

Wavelength 40m 30m 30m

Amplitude 10m 5m 10m

EXPERIMENTAL PROTOCOL
Fibroblasts were seeded onto patterned chips and then incubated in 95% air, 5% CO2 at 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 epifluorescence microscope at 10x and 60x magnification The following measurements were taken:
Cell count Cell length Cell width Cell orientation (relative to pattern)

RESULTS: CELL ALIGNMENT TO PATTERNS

RESULTS: CELL ALIGNMENT TO PATTERNS

Frequency 10% 15% 20% 25% 30% 0% 5%

10 um spaced lines

RESULTS: CELL ALIGNMENT TO PATTERNS

Angle (Degrees)

-90 -85 -80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 More

Frequency 10% 15% 20% 25% 30% 0% 5%

Waves 1

RESULTS: CELL ALIGNMENT TO PATTERNS

Angle (Degrees)

-90 -85 -80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 More

Frequency 10% 15% 20% 25% 30% 0% 5%

Waves 2

RESULTS: CELL ALIGNMENT TO PATTERNS

Angle (Degrees)

-90 -85 -80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 More

Frequency 10% 15% 20% 25% 30% 0% 5%

Waves 3

RESULTS: CELL ALIGNMENT TO PATTERNS

Angle (Degrees)

-90 -85 -80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 More

Frequency 10% 15% 20% 25% 30% 0% 5%

20 um spaced lines

RESULTS: CELL ALIGNMENT TO PATTERNS

Angle (Degrees)

-90 -85 -80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 More

Frequency 10% 15% 20% 25% 30% 0% 5%

No Pattern

RESULTS: CELL ALIGNMENT TO PATTERNS

Angle (Degrees)

-90 -85 -80 -75 -70 -65 -60 -55 -50 -45 -40 -35 -30 -25 -20 -15 -10 -5 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 More

RESULTS: CELL ALIGNMENT TO PATTERNS

RESULTS: CELL ALIGNMENT TO PATTERNS

RESULTS: CELL ALIGNMENT TO PATTERNS

Bottom

Top

RESULTS: CELL ALIGNMENT TO PATTERNS

Bottom

Top

RESULTS: CHANGES IN CELL DIMENSIONS

Cell Length
140.00

120.00

100.00 Cell Length (um)

80.00 24hrs 60.00 48hrs

40.00

20.00

0.00
No Pattern 0% 10um spacing 23.11% 20 um spacing 18.08%: Waves 1 40-10 24.3%: Waves 2 30-5 23.5%: Waves 3 30-10 27.15%:

RESULTS: CHANGES IN CELL DIMENSIONS

Cell Width
25.00

20.00

Cell Width (um)

15.00 24hrs 10.00 48hrs

5.00

0.00
No Pattern 0% 10um spacing 23.11% 20 um spacing 18.08%: Waves 1 40-10 24.3%: Waves 2 30-5 23.5%: Waves 3 30-10 27.15%:

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.

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.