The Effect of Environmental Stiffness on Endothelial Cell and Pericyte Phenotype and Function

By: Adrian Ledesma-MendozaSpecial investigations Seminar

December 15th, 2016

• The microvasculature is the primary site for various exchange.1

• Structure and function vary between tissues.1

• Some of the most common diseases, such as diabetes, have microvasculature repercutions.2

• Microvasculature models often neglect certain elements.

Background

[1] Boron and Boulpaep, Medical Physiology, 2nd Edition, Ch. 20[2] W.T. Cade, Phys Ther. 2008; 88(11). pp. 1322-1335

Boron and Boulpaep, 2nd Edition, Ch. 20

Some Complications…

• Can we develop a model that includes both mechanical and chemical cues?

Lauridsen and Gonzalez. Regen Med. 2014

• PA gels can be conjugated with any protein.3

• Study environmental effects by varying gel stiffness.

• More comprehensive model for disease studies, such as fibrosis.

Polyacrylamide (PA) Gel System

[3] P. Sava’s Thesis. 2016

• Most common interstitial lung disease.

• Affects around 200,000 people in the U.S.

• Fibrosis is produced by an upregulation of extracellular matrix proteins.

• This leads to increased tissue stiffening and eventual tissue failure.

Idiopathic Pulmonary Fibrosis (IPF)

• Most common interstitial lung disease.

• Affects around 200,000 people in the U.S.

• Fibrosis is produced by an upregulation of extracellular matrix proteins.

• This leads to increased tissue stiffening and eventual tissue failure.

Idiopathic Pulmonary Fibrosis (IPF)

Endothelial Cell Phenotype in Response to a Fibrotic Environment

• Grown on different stiffness gels

Collagen I EC

Glass coverslip

Softer gels → Healthy Lung TissueStiffer gels → Fibrotic Lung Tissue

Endothelial Cell Phenotype in Response to a Fibrotic Environment

• Grown on different stiffness gels

• Hypothesis: EC will have increased proliferation on higher stiffness environments.

Collagen I EC

Glass coverslip

Softer gels → Healthy Lung TissueStiffer gels → Fibrotic Lung Tissue

EC on PA: Cell Proliferation1 kPa 5 kPa

8 kPa 20 kPa

EC growth on 1 kPa gels were significantly different at day 3

Scale bar: 100 mPictures taken at Day 1

EC on PA: Cell Proliferation1 kPa 5 kPa

8 kPa 20 kPa

EC growth on 1 kPa gels were significantly different at day 3

Scale bar: 100 mPictures taken at Day 1

EC on PA: Cell Proliferation

EC proliferation was not significantly different between the gels

Control1 kPa5 kPa8 kPa20 kPa

CFSC interpretationHigh MFI → lower cell proliferation

Control is unstained EC

EC on PA: Vessel Formation Potential

5 kPa

8 kPa

- CD34

- DAPI

Sprouting potential decreased as gel stiffness increased

Scale bar: 100 m

1 kPa

20 kPa8 kPa

Scale bar: 100 m

Extracellular Matrix Remodeling by Pericytes

• Treat with TGF-1 and ascorbic acid

Extracellular Matrix Remodeling by Pericytes

Collagen I PC

Glass coverslip

• Treat with TGF-1 and ascorbic acid

• Hypothesis: Environmental stiffness will have an effect on matrix deposition

Extracellular Matrix Remodeling by Pericytes

Collagen I PC

Glass coverslip

Matrix Remodeling by PC: Matrix Imaging

-Laminin-Col I

PC detached from PA. Experimental setup needs revision

TGF- A. Acid

A. Acid PC Media

PC on PAandPC on glass (shown below)

Matrix Remodeling by PC: Matrix Imaging

-Laminin-Col I

PC detached from PA. Experimental setup needs revision

TGF- A. Acid

A. Acid PC Media

Control samples at 7 days

PC on PAandPC on glass (shown below)

Matrix Remodeling by PC: Matrix Imaging

-Laminin-Col I

PC detached from PA. Experimental setup needs revision

TGF- A. Acid

A. Acid PC Media

Control samples at 7 days

PC on PAandPC on glass (shown below)

Conclusion and Future Directions

Investigate the effects of environmental stiffness on EC shape and function

Conclusion and Future Directions

Investigate the effects of environmental stiffness on EC shape and function.✔

Repeat at lower cell density to prevent cell death.

Conclusion and Future Directions

Investigate the effects of environmental stiffness on EC shape and function.✔

Repeat at lower cell density to prevent cell death.

Investigate the effects of environmental stiffness on PC matrix deposition

Conclusion and Future Directions

Investigate the effects of environmental stiffness on EC shape and function.✔

Repeat at lower cell density to prevent cell death.

Investigate the effects of environmental stiffness on PC matrix deposition

FAILED!

Conclusion and Future Directions

Investigate the effects of environmental stiffness on EC shape and function.✔

Repeat at lower cell density to prevent cell death.

Investigate the effects of environmental stiffness on PC matrix deposition✗

Repeat experiment by being more careful managing cells on PA

Conclusion and Future Directions

Investigate the effects of environmental stiffness on EC shape and function.✔

Repeat at lower cell density to prevent cell death.

Investigate the effects of environmental stiffness on PC matrix deposition✗

Repeat experiment by being more careful managing cells on PA

Investigate the effects of PC matrix on EC

Acknowledgements

Gonzalez Lab•Anjelica Gonzalez•Amanda Pellowe•Anand Ramanathan•Holly Lauridsen •Aja Aravamudhan•Rita Matta

Other•Yale SEAS staff •Family•Awesome BME grad students!