Understanding Keratoconus

Orange County Vision Symposium Cristina Kenney, MD, PhD

Gavin Herbert Eye InstituteUniversity of California, Irvine

Saturday June 26, 2010

No Financial Disclosure

• Three Major Pathological Features of KC

1. Corneal Thinning

2. Oxidative Damage

3. Anterior Corneal Instability

Outline

• Numerous Molecular and Genetic Studies Conducted on KC

Are These Features Related to Each Other?

Pathological Featuresof Keratoconus

• Corneal Thinning is a Hallmark of KC

• KC corneas have:

– Increased degradative enzyme activities

– Decreased levels of enzyme inhibitors

Degradative EnzymesCollagen/Matrix Produced

Thin Cornea

Degradative Enzymes

Collagen/Matrix Produced

Epithelium

Bowman’s Layer

Stroma

Descemet’s membrane

Endothelium

Epithelium

Endothelium

Keratoconus Cornea

Apoptosis

Thinning of cornea

Loss of Bowman’slayer

Outline

• Pathological Features of KC

1. Corneal Thinning

2. Oxidative Damage

3. Anterior Corneal Instability

Schematic of the CellUVB Mechanical

Lipid Peroxidation

INCREASEDPeroxynitrites

AntioxidantAntioxidantEnzymesEnzymes

Oxidative Damage

Nitric Oxide

ROS/RNS

INCREASEDCytotoxic aldehydes

ACTIVATEDEGRADATIVE ENZYMES

Mitochondria

Schematic of the Cell

UVB Mechanical

Lipid Peroxidation

INCREASEDPeroxynitrites

AntioxidantAntioxidantEnzymesEnzymes

Oxidative Damage

Nitric

Oxide

ROS/RNS

INCREASEDCytotoxic aldehydes

ACTIVATEDEGRADATIVE ENZYMES

Mitochondria

Schematic of the Cell

DNA - Nuclear vs Mitochondrial

• Mother and Father• Double helix

• Mother only• Circular

16569/0

D-Loop

16srRNA

Cyt b

T

END6

ND5

ND2ND4

ND4L

R

G

KD

S

W

OL

L

V

ND1

MIQ

COIIICOII

COI

ATPase6

ATPase8

ND3

LSH

^

12srRNA

F

P

CY

NA

mtDNA is

• Particularly susceptible to damage

• Repair mechanism is poor

Question #1:

• Extracted DNA from normal & KC corneas

• Analyzed mtDNA

• Atilano et al 2005 Invest Ophthal Vis Sci 46:1256-63.

Are the mitochondria altered in KC corneas?

Results

Normal Keratoconus0.0

2.5

5.0

7.5

10.0

12.5

15.0

Normal

Keratoconus

# o

f b

and

s

Normal Keratoconus0.0

2.5

5.0

7.5

10.0

12.5

15.0

Normal

Keratoconus

# o

f b

and

s

mtDNA defects per individual

NL = 4.3 ± 2.7 KC = 7.4 ± 3.76P<0.04

Conclusions Of This Study

• mtDNA from KC corneas are more damaged than the normal corneas

Importance of Our Findings

• Defects in the mtDNA can cause:

– Increased ROS/RNS Production

– Less Energy Produced

– Loss of Function

– Increased Cell Death

Healthy Cell - Numerous mitochondria

Nucleus Mitochondria = Energy (battery)

Cell with mitochondrial damagehas less energy produced

Cell lacking energy start dying

Healthy Cell - Numerous mitochondria

Nucleus Mitochondria = Energy (battery)

Cell with mitochondrial damagehas less energy produced

Cell lacking energy start dying

Question #2:

• Cultured Normal & KC Corneal Cells

• Stressed Cells by Two Methods

– Hydrogen peroxide (H2O2)

– Lowered pH to increase metabolic demand

Do the KC cells behave similar to cells from normal corneas?

Under Identical Conditions

• KC Cells Had:

– Higher ROS/RNS production

– Increased mtDNA damage

– Decreased mitochondrial function

– Increased cell death

Conclusions

• Keratoconus cells are overly sensitive to stressors

– Poorly fit contact lenses

– Vigorous eye rubbing

– UV light

– Allergies

• Can not process the ROS produced by these

stressors as efficiently as normal corneal cells

Oxidative Stress in Keratoconus

These findings support our overall

hypothesis that Oxidative Damage

contributes to keratoconus pathology

M o d i f ie d f r o m Y a k e s & V a n H o u t e n ( P N A S 1 9 9 7 )

A . N o r m a l C e l l

O x i d a t i v e

S t r e s s

A n t i o x i d a n t e n z y m e s

L i p i d p e r o x i d a t i o n e n z y m e s

E l i m i n a t i o n o f R O S

R O S C o m p o u n d s

M i n i m a l m t D N A

d a m a g e

N o r m a l c e l l

f u n c t i o n

AtopyEye rubbingStretchingUVB light

B. Keratoconus Cell

ROS Compounds

mtDNA damage

OXPHOS

ROS Production

Abnormal cellfunction

Corneal thinning

Abnormal antioxidant enzymesLipid peroxidation enzymes

Accumulation of ROS

AtopyEye rubbingStretchingUVB lightGenetic defects

OxidativeStress

Enzymes ActivityTissue Degradation

Outline

• Review Pathological Features of KC

– Corneal Thinning

– Oxidative Damage

– Anterior Corneal Instability

• Are These Features Related to Each Other?

Slippage Theory – Lamellae

• Normal Corneal Stroma

– Layers or lamellae

– Very organized

– Layers are interconnected and do not slip

• KC Corneal Stroma

– Layer or lamellar - Uneven distribution

– Not organized– Meek et al, 2005; Bergmanson et al 2009

Slippage Theory – Fibrils

• Normal Corneas

– “Anchoring” fibrils that insert

transversely for 120 μm into Bowman’s

layer

• KC Corneas

– Lack these anchoring fibrils• Morishige et al, 2007

Slippage Theory

Normal Cornea

Epithelium

Descemet’s membrane

Stroma

Bowman’s layer

Endothelium

Transverse “Anchoring” Fibrils

Slippage Theory

Epithelium

Endothelium

Loss of Transverse“Anchoring” Fibrils

Lamellar Slippage Lamellar Slippage

Keratoconus Cornea

OcularPressure

Consequences

• Lamellae and anchoring fibrils

– Function to maintain the corneal shape

– If you lose the lamellae and/or fibrils you can

get slippage, stretching, and warpage • (Bron et al, 2001; Muller et al, 2001)

• Slippage Creates An Unstable Substrate

Potential Sites of Interaction

1 2

3

Ways to Block Enzyme Activities

• Cultured cells were treated with nicotine

• Nicotine increased the degradative enzyme activities

– MMP-2, MMP-9, MMP-8, gelatinase

• KC Patients Should Not Smoke

– Jacob-Ferreira et al Eur J Pharmacol. 2010 10;627(1-3):216-22 – Sørensen et al Regen. 2009 17(3):347-53

Blocking Enzyme Activities

• Collected tears & cells from glaucoma patients

• Latanoprost (Xalatan-prostaglandin analogue)

– Increased MMPs and Decreased TIMP-1

• If a KC patient also has glaucoma they probably should not use this family of glaucoma drops

• May also be true for patients after LASIK – Honda et al Arch Ophthalmol. 2010;128:466-71.

Future Studies

• Need to identify inhibitors that can block degradative enzymes

– Doxycycline

– Epicatechins

– Others

Blocking ROS/RNS Formation

• KC cells do not process stress as well as normal cells

• KC patients would benefit by protecting their eyes from environmental sources of ROS/RNS or any source of inflammation

– UV light

– Atopy/allergies

– Vigorous eye rubbing

– Poorly fit contact lenses

Recommendations for KC Patients

• Provide UV Protection

– Glasses or contact lenses

– UV light can cause formation of ROS/RNS which KC patients can not process normally

• Contact Lenses

– Proper fit is critical

– Improperly fit contacts can lead to increased inflammatory cytokines and ROS formation

Recommendations for KC Patients

• Lubricate Eyes

– Preservative free artificial tears

– Want eyes to be as comfortable as possible

• Atopy or Allergies

– Control with medications

– Anti-allergy medications

– Non-steroidal anti-inflammatory drops

– Allergies can lead to rubbing which causes mechanical stress on the cornea

Recommendations for KC Patients

• Diet with High Levels of Antioxidants - Similar to AMD diet

– Green Leafy Vegetables

• Spinach, Kale, Green Beans, Broccoli

– All Vegetables with Color

• Tomatoes, Peppers

Supplemental Antioxidants That Benefit the Retina in AMD Patients

• Omega-3 Fatty Acids

• Vitamin C 500 mg

• Vitamin E 400 IU

• Zinc 80 mg

• Copper 2 mg

• Beta-carotene 15 mg

Blocking Corneal Instability

• Stabilize the Corneal Substrate

– Cofactors - Promote Healthy Collagen Formation

• Ascorbic Acid

• Manganese

• Copper

• Lysine

• Proline

– Intacs

– Cross-linking Treatments

Potential Areas of Intervention

Acknowledgements:

• National Keratoconus Foundation

• NDRI

• Douglas Wallace, PhD• Pinar Coskun, PhD• Donald Brown, PhD• Nitin Udar, PhD• Anthony Nesburn, MD• Ezra Maguen, MD• Marilyn Chwa, MS• Shari Atilano, MS

Supported by:

• NIH EY06807

• Jane & Norman Neely Foundation

• Discovery Eye Foundation

• Schoellerman Charitable Trust

• Skirball Foundation

• Guenther Foundation

THANK YOU