ALTERING ANGIOGENESIS

IN VITRO

Dr. Alvin Yeh Department of

Biomedical Engineering

Dr. Arne LekvenDepartment of Biology

Josh Bergerson

Normangee High School

Components of Engineering

BiomedicalGenetic

Optics

Tissue

Mechanics

Computer

Faculty Members

Dr. Alvin Yeh• Ph.D., Chemistry,

University of California, Berkeley, 2000

• B.S.E., Chemical Engineering, University of Michigan, 1993

Dr. Arne Lekven• B.A., 1989, UC San Diego,

Animal Physiology. • Ph.D., 1997, UCLA,

Molecular, Cell and Developmental Biology

Lab Group Members

• M.Sc. University of Edinburgh (2008)

• Gene expression in zebrafish

Holly Gibbs

• M.S. Shanghai Jiao Tong University (2002)

• Engineered tissue scaffold developmentYuqiang

“Bob” Bai

• M.S. University of Colorado, Boulder (2005)

• Angiogenesis modeling and manipulationPo-Feng

Lee

Angiogenesis

New blood vessel (capillary) formation

Important in tissue growth & repair

Excessive cancer

Insufficient stroke

Angiogenic Process

ECs detach from wall

Degrade & penetrate basal lamina

Invade surrounding ECM

Extracellular Matrix

Scaffold; structural support

Adhesive contact sites

Mechanical & biochemical signals

Research Question

What effect does changing the collagen fiber stiffness

and thickness have on angiogenic patterns in vitro?

angiogenesis in

diseases

vascularzing

engineered tissue

In Vitro Angiogenesis Model

Endothelial cell

monolayer

Collagen gel preparation

Serum–free medium

Sandwich Modeling

2 possible matrices

Varying fiber stiffness

Observe angiogenic

growth

Fiber Thickness

Polymerizing collagen at varying temperatures

Observe angiogenic patterns in matrix

Data Acquisition

TPF, SHG & Light microscopy

SHG detector

TPF detectorUltrafast laser

Objective

Two-Photon Microscopy Used to create 3D

images from optical sections

Detects sample’s fluorescence (cell/GFP/etc)

Wavelength ~ 405nm

TPF-3D TPF-2D

Second Harmonic Generation (SHG)

Sample mixes 2 photons Detects collagen; crystal,

repeating structure Not measuring

fluorescence Wavelength ~ 480 nm

Second Harmonic Generation (SHG)

E2

E1

hvin

hvin

Virtual States

SHG TPF+SHG

TPF & SHG Imaging depth ~

500um Laser Bandwidth

~ 133nm centered at 800nm

Ability to image living cells

10 femtosecond pulse laser (1x10-

14 s) PMT detectors

Data Analysis

Morphology Cellular Growth

Lumen Development

1 mg/ml 2.5 mg/ml

Brightfield TPF+SHG

Possible Classroom Application

Physics

:

•Optics

•Wave properties

•Mechanics

Chemistry:

•Electromagnetic Spectrum

•Behavior of Electrons

•Biochemistry

Acknowledgements

TAMU E3 RET Program National Science Foundation Nuclear Power Institute Dr. Alvin Yeh Research Group:

Tissue Microscopy Lab Dr. Arne Lekven Lab Group

Conventional Microscopy

Non-laser light source

Snapshot rather than scanning

Imaging depth ~20um

Single Photon Microscopy Imaging depth

~100um Continuous laser

excitation Pinhole allows for

optical sectioning Photobleaching of

fluorescent probes

Tracking Gene Expression Phenotypic expression of brain

development genes is know but specifics are not (wnt1, pax2, fgf8)

Genes can be tracked by tagging with fluorescent proteins

Allows detection of gene being “turned on” during development

Grown on collagen or fibrin scaffold

Engineered Tissue Scaffolds

skin blood vessels

tendon ligament

soft connective

tissue

Spectral Two-Photon Microscopy

16 PMT spectrometer

Computational linear unmixing

Spectral Two-Photon Microscopy To detect gene

expression Simultaneous

detection of multiple fluorescent proteins

Real-time study of live embryonic development

Second Harmonic Generation (SHG)

Detects collagen Requires crystal,

repeating structure Not measuring

fluorescence

Optical Coherence Microscopy (OCM)

Collects light reflected from sample (morphology)

Collects data via spectral detector (significant power loss)

Optical Coherence Microscopy (OCM) Detects fibrin, collagen,

and cell Factor out collagen and

cell through TPF & SHG Observe growth of cell

scaffold under various conditions

Research Goals

Gene Expression

3D data acquisition over time

Visualizing gene

expression

Engineered Tissue

Microscopic; ECM and

cells

Macroscopic;

mechanical properties

Angiogenesis

ECM interaction Environmen

t influences patterns

Wnt1 Gene Expression

Mark Feltner, M.A. Skyline High School Dallas, Texas

Laser’s 2-photon emission, in conjunction with fluorescent proteins, is providing us with more detailed imaging of developing embryos.

Several of the genes discussed are oncogenes – they can, under some circumstances, initiate cancerous growth.

(Q: these include which: wnt 1, wnt 8 (all wnt’s??) spt, pax 2…others?)

Each gene is involved in early brain development. The sp5 gene expression, for instance, is involved in development of the midbrain, hindbrain and spinal cord, but not the forebrain.

(Holly’s schematic here – circular, yellow. NB p 11. Shows mb/hb/sc/fb regions in yolked embryo)

wnt1 pax2a

fgf8

Multiple genes work together in the same place at the same time

In early zebrafish emryos, the mesoderm and endoderm are initially mixed.

They differentiate.

But these are ‘just’ fish, right? How is this relevant to us?

In every vertebrate embryo, there is always a midbrain-hindbrain boundary. This goes for all mammalian species, including us, the mighty Homo sapiens.

In fact, this diagram helps illustrate the commonalities of gene expression that all vertebrate species share.

To recap: Before you can actually see a difference in cell formation, the cells are already expressing the various genes that will cause them to differentiate.

The wnt1 gene appears to be conserved across multiple vertebrate species, including humans.

Thus: understand zerbrafish early brain development, and we can better understand mammalian gene expression of brain development.

This is huge.

Currently, we know what expression of wnt1 gene does, but nobody knows the mechanism of how this gene gets turned on.

This is one question that we hope to answer. Of course, there are many more.