PROLIFERATIVE VITREORETINOPATHY: ARRAY OR DISARRAY

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PROLIFERATIVE VITREORETINOPATHY ARRAY OR DISARRAY
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OUTLINE

• Introduction
• Structure of Retina
• Proliferative Vitreoretinopathy
• cDNA macroarrays
• Real time PCR
• cDNA microarrays
• Future Work

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ANATOMY OF THE EYE
http//webvision.med.utah.edu/sretina.html
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CROSS-SECTION OF RETINA
CHOROID
http//webvision.med.utah.edu/sretina.html
VITREOUS
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FUNCTIONS OF RPE
choroid

• Barrier epithelium
• Reduce light scattering
• Visual cycle
• Phagocytosis
• Keeps the retina attached

vitreous
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CHARACTERISTICS OF RPE

• Normally non-dividing
• But can divide
• tissue culture
• pathological conditions

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PROLIFERATIVE VITREORETINOPATHY (PVR)

• Tear/damage in retina
• RPE cells lose contact inhibition
• Cells gain access to vitreous
• Proliferate
• Make extracellular matrix (ECM)

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PVR (CONTD.)

• RPE cells attach to collagen fibers and other
  components of the ECM
• Epiretinal membranes formed
• Migration/attachment - tangential force on the
  retina
• Anterior and posterior vitreous cortex contracts
• Retinal detachment

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RISK FACTORS FOR PVR

• Retinal holes, retinal breaks or retinal tears
  (eye trauma )
• RPE cells in vitreous
• Breakdown of blood-retinal barrier and/or
  inflammation
• Mechanical forces on retina - traction
• Hereditary abnormalities of peripheral retina
  -myopia

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FORMATION OF EPIRETINAL MEMBRANES
Liou G, unpublished results
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CLINICAL IMPORTANCE

• In the US and Worldwide 5-10 of retinal
  detachment is due to PVR
• Surgical treatment not very effective
• Past approach in the treatment of PVR has been
  disappointing
• Need to develop new therapeutic agents

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EPITHELIAL-MESENCHYMAL TRANSFORMATION (EMT)

• Transformation occurs in development and disease
• Epithelial cells undergoing EMT
• lose cell-cell and cell-basement membrane
  interactions
• fibroblast-like morphology
• become mobile
• proliferative and invasive
• Very similar to changes that occur during
  tumor formation

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VITREOUS HUMOR AND PVR

• Vitreous shown to contribute to EMT in cultured
  RPE
• RPE cells with fibroblast morphology cause
  contraction of the collagen gel in a model system.

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Effect of Vitreous on the Morphology of RPE Cells
B
A
CONTROL
25 VITREOUS
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VITREOUS

• Gel
• Collagen fibers in hyaluronic acid matrix
• Collagen II
• some collagen V/XI, VI, IX
• Also contains variety of other components
• transferrin, albumin
• chemotactic factors, growth factors and
  morphogenic factors, matrix metalloproteinases
  etc

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WHAT DO WE KNOW ABOUT PVR-MOLECULAR LEVEL?

• HOW SHOULD WE APPROACH THIS PROBLEM?
• Detachment
• Motility
• Invasiveness
• ECM formation
• Contraction

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APPROACHES TO IDENTIFICATION OF MOLECULAR LESIONS

• Individual gene approach
• RT-PCR
• Northern blots
• Western blots
• Global approach
• cDNA macroarrays
• cDNA microarrays

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MACROARRAYS

• Clontech
• Postively charged nylon membrane (588 double
  spots)
• Isotope (p-32)
• Expose to film and develop (1-10 days)

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cDNA MACROARRAY (10 day exposure)
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Change in mPGES and MMP-14 RNA Expression after
Incubation of RPE cells with Vitreous
PGES
PGES
MMP-14
MMP-14
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QUICK CHECK FOR DNA CONTAMINATION
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REAL TIME PCR VALIDATION
Real-time PCR analyses RPLPO and PGES
Control cells
Vitreous-treated cells
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OTHER GENES VALIDATED

• Alpha-5 integrin
• MMP-14
• COX-2

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LIMITATIONS

• Why switch from membranes to glass slides?
• Some limitations of membrane arrays
• Expensive
• Fewer genes represented
• Control and experimental samples on different
  membranes
• Bleeding from adjacent spots very common problem
• High background

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GLASS SLIDE ARRAYS or MICROARRAYS

• Probe is tethered to an immobile surface, such as
  glass
• Glass durable, non-porous, allows covalent
  attachment of probe
• Probe spotted by pins or capillary tubes
• Low background fluorescence

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GLASS SLIDE ARRAYS or MICROARRAYS (CONTD.)

• Sample hybridized to array
• Detected by fluorescence
• At least two fluorochromes used, cy3 and cy5 i.e,
  simultaneous hybridization
• Massive amounts of data generated (data mining)

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DISADVANTAGES

• Limitations of microarrays
• Large amounts of starting RNA required
• Stability problems with light exposure
• Need laser scanner
• Current location of scanner

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PREPARATION OF TARGET PROBE
DIRECT and INDIRECT LABELING
Direct incorporation of cy3/cy5 labeled dCTP
during RT, i.e enzymatic incorporation
RT using aminoallyl dUTP (amine modified analog
of dUTP) Post labeling with cy3/cy5 coupled to
NHS ester
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cDNA AMPLIFICATION

• At least 100ug of total RNA needed
• Amplification necessary
• Eberwine method using the T7 system

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DISADVANTAGES OF PCR LABELING OVER T7

• Skewing of population is more because it is
  logarithmic over linear skewing using T7
• As a result of logarithmic skewing some RNAs
  might never be represented in the PCR system

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AMBION T7 AMPLIFICATION
RT
RT
First strand cDNA synthesis
pol
X?X?X?
Rnase H digestion products - primer
Double strand cDNA
pol
pol
pol
pol
Copied strands are anti-sense to original mRNA
aRNA
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CYSCRIBE DIRECT LABELING PROTOCOL

• Take 1ug aRNA from T7 amplification
• RT using cy3/cy5 labeled dCTP
• Purify and concentrate
• Hybridize 14-16hrs in 37C incubator
• Wash 3 times with high and low salt
• Scan using Packard Scanner at RMH

INCORPORATION OF DYES DURING RT
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POINTS TO REMEMBER

• Control and experimental sample
• Labeled with two fluors
• Hybridized to same microarray
• False color palette
• Red-cy3, green cy-5
• Yellow-equal representation of cDNA in both
  samples

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CYSCRIBE DIRECT LABELING
1.7k gene array
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CYSCRIBE DIRECT LABELING
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AMBION POST LABELING PROTOCOL

• Take 1ug aRNA from T7 amplification
• RT using Ambion amino allyl kit (aadUTP)
• Couple with NHS form of cy3/cy5
• Purify and concentrate
• Hybridize 14-16hrs in 37C incubator
• Wash 3 times with high and low salt
• Scan using Packard Scanner at RMH

INCORPORATION OF DYES AFTER RT
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AMBION POST LABELING
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AMBION POST LABELING
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COMPARISON OF DIRECT AND POST LABELING

• Very similar over all results

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UPSCALING TO 20K GENE ARRAYS

• University of Toronto (OCI)
• 20000 genes double spotted on two slides

Control epithelial cells Experimental
mesenchymal cells ( vitreous) Time course 0-48
hours Labeling method direct
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20K GENE ARRAY
20K GENE ARRAY SLIDE A
20K GENE ARRAY SLIDE B
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Vitreous
6hr
12hr
24hr
48hr
Epithelial Cuboidal Polarized Adherent Non-motile
Fibroblastic Elongated Non-polarized Separated Mot
ile
Loss of shape
Overgrowth
Signals Receptors Transcription factors
Cytoskeletal Changes Actin - keratin
Intercellular Adherent Protein changes ECM-integri
n changes Motility proteins
Possible Changes
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ADHESION/SKELETAL PROTEINS IN FIRST 20K GENE ARRAY
Vitreous
6hr
12hr
24hr
48hr
Epithelial Cuboidal Polarized Adherent Non-motile
Fibroblastic Elongated Non-polarized Separated Mot
ile
Loss of shape
Overgrowth
Signals Receptors Transcription factors
Cytoskeletal Changes Actin - keratin
Intercellular Adherent Protein changes ECM-integri
n changes Motility proteins

• NR CAM
• Alpha 3 integrin (proteome)
• Zyxin (adherens junction of RPE)
• Contactin
• Cadherin 8
• Proteoglycan-2
• Myosin SMA
• Alpha 2 actin
• Gamma 2 actin

• Alpha 5 integrin (proteome)
• Restin (int filament)
• Lamin B receptor
• Transmembrane protease ser-4 (in tumor
  progression)

Adhesion proteins UP at 24 hours
Adhesion proteins DOWN at 24 hours
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SIGNALING PROTEINS
Vitreous
6hr
12hr
24hr
48hr
Epithelial Cuboidal Polarized Adherent Non-motile
Fibroblastic Elongated Non-polarized Separated Mot
ile
Loss of shape
Overgrowth
Signals Receptors Transcription factors
Cytoskeletal Changes Actin - keratin
Intercellular Adherent Protein changes ECM-integri
n changes Motility proteins

• Citron (rho-interacting ser/thr kinase)
• SFRS protein kinase
• Endothelin receptor
• Phospholipase I
• ZFP transcription factor
• VASP (signals to actin)

• FGF receptor 3
• FGF receptor 2
• IGF binding protein 3
• Glutamate receptor

Signaling proteins UP at 24 hours
Signaling proteins DOWN at 24 hours
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WHAT NOW?

• Arrays at various time points
• Both direct and post-labeling
• Repeat with different donors and vitreii
• VALIDATE!!!
• Real time PCR
• Western blots
• si RNA
• Elucidate interesting pathways
• Data Analysis (more sophisticated analysis
  programs)
• Discover all the road blocks and pitfalls along
  those pathways?

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SO WHAT???

• ELUCIDATION OF SIGNALING PATHWAYS AND MOLECULAR
  THERAPEUTICS

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ACKNOWLEDGEMENTS

• Drs. Hunts
• Sunil
• Walter
• Ola
• Ren