Dr. Juan B. Kouri Flores

Departamento de Infectómica y Patogénesis Molecular

Centro de Investigación y Estudios Avanzados

(CINVESTAV)

Mexico

Site and severity of OA

Systemic factors Mechanical factors

Arden N, Best Practice & Research Clinical Rheumatology

2006 Vol. 20, No. 1, pp. 3–25

Felson DT. Ann Intern Med. 2000 Oct 17;133(8):635-46,

Susceptibility to

damage in the

joint, fault repair

and OA appears

Age

Gender

Menopause

Genetics

Nutrition

Bone Density

Obesity

Surgery

Trauma

Joint deformations

High impact sports

Repeated Loading in

joint

CARTILAGE FROM HUMAN

CARTILAGE FROM HUMAN vs RATS

OA

NormalCARTILAGE FROM RAT

HUMAN

Microscopical Study

OANORMAL

RAT

Cellular Aggregates From Human OA Cartilage

Phenotypic variability of OA chondrocyteKouri et al.1996

Phenotypic variability chondrocyte

within an OA rat Modelo

Normal OA 5 days OA 10 days

Predominant Ultrastructural Pattern

Normal OA 5 days OA 10 days

OA 20 days OA 45 days OA 60 days

Kourí, et al., 2000

NE

N

Extracelular Matrix

OA Rat Model

Safranin O Staining

3 6 8 10

Time (days)

S

OA

CHONDROPTOSISINJURY REPARATIVE STAGE DEGRADATIVE STAGE

HYPOTHESIS

Normal Caspase 3-LC3Perlecan MMP-3

"Activation" and "Transdifferentiation" of the chondrocyte phenotype

Kouri JB, Lavalle C. 2006Roach T. Aigner T Kouri J.B. 2004

Almonte et al. 2010

Degradative Phase

MMPS

Tissue reparative phase

synthesis of perlecan,

biglycan and decorin

( Matrix Fragments )

Micro-repetitive

mechanical cartilage damage

Generation of inflammation

Osteoarthritis Pathogenesis

MMPS

ECM degradation and Chondroptosis

Chondrocytes

IL-1, TNF-α

Goldring MB. (2007). J Cell Physiol.Dec;213(3):626.

Kouri JB, Lavalle C. (2006). Histol Histopathol 21(7):793.

More cartilage damage

Generation of inflammation

Damaged Joint

ARTICULAR CARTILAGE

Cytokines balance

NORMAL OA

KEEPING EQUILIBRIUM EQUILIBRIUM IS LOST

EXTRACELLULAR MATRIX IS LOST,THE NUMBER OF CHONDROCYTES ARE REDUCED AND THEIR

FUNCTIONS ARE MODIFIED

Experimental Groups : Normal, Normal with excercise, OA and Sham

CONFOCAL ANALYSIS

IL 1-β, IL-10, TGF-β1,

Wistar Rats ♂130 – 150 g

CONFOCAL ANALYSIS

(% of positives cells)

EACH EXPERIMENT WAS FROM 9 SLIDES, 27 SECTIONS, 81 FIELDS

Each sections3 RANDOM FIELDS

NO-PARAMETRIC ANOVA STATISTICAL

TEST

OA

IL-1β

NE

Normal cartilage 3 td 6 td 10 td

A

0

20

40

60

80

100

Normal cartilage

3 td 6 td 10 td

IL-1β

positiv

e c

ells

(%

)

OA

NE

**

**

**

**# ## ## ## #

B

*vs Normal

# vs OA

TGF-β1A

OA

NE

N 3 td 6 td 10 td

0

20

40

60

80

100

N 3 td 6 td 10 td

TG

F-β

1 p

ositiv

e c

ells

(%

)

OA

NE

*

*

*

*

*

*

# ## ## ## #

*

*

# # # # ####

# ## ## ## #

B

*vs Normal

# vs OA

OA

NE

N 3 td 6 td 10 td

IL-10A

0

20

40

60

80

100

N 3 td 6 td 10 td

IL-1

0 p

ositiv

e c

ells

(%

)

OA

NE

*

*

*

*

# ## ## ## #

*

*

# # # # ####

# ## ## ## #

B

*vs Normal

# vs OA

n=1

X 10

X 10

n=1

n=3

Pool of cartilage

Protein extraction for

each experimental group.

Western blot for IL-

Statistical Western Blott

Analysis

X 10

n=1

n=1

Statistical analysis

(ANOVA)

Wistar rats ♂130 – 150 g

X 10 n=3Western blot for IL-

1β, TNF-α, TGF-β and

IL-10.

Quantification by

densitometry with the

Image J software

IL-1β

TNF-α

0

0.3

0.6

0.9

1.2

Normal cartilage

3 td 6 td 8 td 10 td

Rela

tive

expre

ssio

n(I

L-

1β

/actin)

OA

NE

OA

NE

Actin

Normal

cartilage 3 td 6 td 8 td 10 td

***

*

# ## ## ## #**####

A

BTNF-α

0

0.1

0.2

0.3

0.4

0.5

0.6

Normal cartilage

3 td 6 td 8 td 10 td

Rela

tive

expre

ssio

n(T

NF

-

α/a

ctin)

OA

NE

OA

NE

Actin

Normal

cartilage 3 td 6 td 8 td 10 td**

*

# ## ## ## #

*

#### **

*vs Normal

# vs OA

0.0

1.0

2.0

3.0

Normal cartilage

3 td 6 td 8 td 10 td

Rela

tive

expre

ssio

n(T

GF

-β

/actin)

OA

NE

OA

NE

Actin

Normal

cartilage 3 td 6 td 8 td 10 td**

* ***

*

TGF-β1A

B

0.0

1.0

2.0

3.0

Normal cartilage

3 td 6 td 8 td 10 td

Rela

tive

expre

ssio

n(I

L-

10/a

ctin)

OA

NE

OA

NE

Actin

Normal

cartilage 3 td 6 td 8 td 10 td** *

*

** ####

####

####

IL-10

B

*vs Normal

# vs OA

RAT HUMAN

Normal OA

E

GRP78

PDIA1

HSP7CALBU

PDIA3

ATPBNUCB2

RINI SPRC ENOG

ACTBGUAD

KCRBNAGAB

ACTGACTBSPRC

ENO A

EF1GOAT

CAPG

ACY1A

DDAH1

kDa

75

50

37

100

150

250

GDIB

ADK

ARSB

GRP78

PDIA1

HSP7CALBU

PDIA3

ATPBNUCB2

RINI SPRC ENOG

ACTBGUAD

KCRBNAGAB

ACTGACTBSPRC

ENO A

EF1GOAT

CAPG

ACY1A

DDAH1

kDa

75

50

37

100

150

250

GDIB

ADK

ARSB

Expression of Latexin in the articular cartilage

Pérez et al, 2010. Proteome Science

F

COL1A1

ANXA5

COL2A1

MDHC

DDAH1

CLIC6

CATBDDAH2

PA1B2

LXN RSSA

THTM

ERP29

PEBP1

PRDX2

GDIB

MLE1

LGULTCTP

37

20

10

1433Z 1433T

TPM1

TPM4

1433E

4 5 6 7 pI

PGAM-B

COL1A1

ANXA5

COL2A1

MDHC

DDAH1

CLIC6

CATBDDAH2

PA1B2

LXN RSSA

THTM

ERP29

PEBP1

PRDX2

GDIB

MLE1

LGULTCTP

37

20

10

1433Z 1433T

TPM1

TPM4

1433E

4 5 6 7 pI

PGAM-B

Proteins bidimensional map normal rat AC (SDS-10% acrylamide, pH 4-7

nonlinear, silver staining). Protein names are indicated according to the Pilot

Protein database

Anti-latexin/FITC: A, B-chondrocytes SZ(N); C, D (OA cartilage "clones" cell

SZ); E, F (OA cartilage, MZ. . Confocal.Microscopy

COLLABORATORS

ASSISTANCES:Magdalena Miranda

Raymundo Cruz

Sirenia González

STUDENTS:Mariel Rojas

Karim Abbud

Madaí Gómez

Adriana Alvarado

Norma Capín

Aurora Scott

Elizabeth Pérez

Consuelo Zermeño

Guadalupe Jiménez

INR

IMSS

ISSSTE

UNAM

INN

INSTITUTIONS

CINVESTAVSirenia González

Clara Castelán

Guadalupe Jiménez

Elena C. Gonzáles

Matilde Vázquez

Maylin Almonte

América Martínez

Nancy Torres

Josefina Arellano

Leticia Bañuelos

David Solís

Moisés Cabrera

Cecilia Espinosa

Rafael Jijón

THANK YOU

CINVESTAVMarco Vega

Fidel de la Cruz

Rogelio Fragoso

Fernando Navarro

Vianey Ortiz

Our results suggest that during OA progression chondrocytes undergo dramatic phenotypic changes and

display signaling transduction machinery capable of inducing its own morpho-functional changes. In early

OA, chondrocytes increase ER and Golgi in order to synthesize proteins required for ECM reparation.

However, when the repair capacity is overwhelmed, chondrocytes begin the synthesis of catabolic molecules

CONCLUSION

like IL-1β, IL-6, TNF-α that stimulate an inflammatory process and degradation of ECM by metalloproteases

like MMP3 and MMP-13. Furthermore, the decrease of anti-inflammatory molecules in OA could be involved

at the beginning of the disease. Finally, when chondrocytes loose their reparative capacity, execute its own

cell death program that includes both autophagy and apoptosis, which called chondroptosis.