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Uso de Modelos In Vitro e In Vivo en el

Diseo de Nuevas Estrategias Teraputicas


Dirigidas a Blancos Moleculares En
Enfermedades Cardiovasculares.
SILVIA S. PIERANGELI, Ph.D.
Professor
What is the Antiphospholipid Antibody Syndrome ?
An acquired autoimmune thrombophilia,
characterized by:
a) vascular thrombosis.
b) recurrent pregnancy losses.
c) thrombocytopenia.
d) laboratory evidence for:
-antibodies against phospholipids or
phospholipid-binding protein cofactors.
APS morbidity
APS is the most common cause of acquired thrombophilia.
Prevalence in general population: 2-4%
15-20% of all DVT with or without PE.
1/3 of new strokes in patients < 50 years age.
10-15% women with recurrent pregnancy losses.
APS: significant proportion of thromboembolic disease and
pregnancy loss in SLE.
APL Abs present in 30-40% SLE. One third of those patients
have clinical manifestations of APS.
aCL positivity may precede a more severe form of SLE.

Multiple Strokes
in a Young Woman
(Brain MRI)
Occlusion of Right Middle Cerebral Artery
In a 3 Years Old Child with
Severe Headache and Hemiparesis
With aCL Antibodies +
Digital Necrosis and Gangrene
Diagnostic Tests

Anticardiolipin Test
Lupus Anticoagulant Test
ANTI-CARDIOLIPIN TEST
Advantages
Overwhelming majority of APS patients are anti cardiolipin positive
Test can be performed reproducibly.
Clinicians and laboratories generally familiar with units of
measurement.


Disadvantages
Relatively nonspecific (particularly low positive, IgM positive).
Intra-laboratory and Inter-laboratory variability.
Problems with false positive results: aCL positive in a wide variety of
infectious diseases and in non-APS related autoimmune diseases.

Predictive value of IgG aCL for thrombosis in
patients with SLE (Escalante et al)
IgG aCL levels below 21.4 = probability of
thrombosis 0.07

IgG aCL levels >21.4 and < 65.0 GPL =
probability of thrombosis 0.20

IgG aCL levels >65.1 GPL units = probability
of thrombosis 0.75
Anti-
2
glycoprotein I
More specific than anticardiolipin test for diagnosis of
Antiphospholipid Syndrome (but not 100% specific)
Not as sensitive as anticardiolipin test (70-90% sensitivity)
Efforts of standardization continuing
Useful in diagnosis of doubtful cases of APS. Some APS
patients negative for aCL and positive for anti-

2
GPI.

APhL

ELISA - Principle
Based on observation that antiphospholipid antibodies cross-
react with negatively charged phospholipids but syphilis and
other infectious diseases sera largely limited to cardiolipin
binding (no cross-reactivity)
Construction of a kit with negatively charged phospholipids
might eliminate non-specific binding.
APhL

ELISA kit
Antigen composed fo mixture of phospholipids
instead of cardiolipin
Sensitivity of APS (greater than 90%)
More specific than anticardiolipin test and at least
as specific (or more) compared to anti-
2
GPI
Incorporation of an in-house positive control
Can be utilized for first line testing, and certainly
important in confirmation of APS
Erkan and Lockshin, Rheum Dis Clin North Am
2006;32:129-48
Lim et al. JAMA 2006;295:1050-1057
Vascular Thrombosis
prevention
Asymptomatic aPL No treatment or ASA?
Venous thrombosis Warfarin INR 2.0-3.0
Arterial thrombosis Warfarin INR 3.0
Recurrent thrombosis Warfarin INR 3.0-4.0+ASA
CAPS Anticoagulation +
corticosteroid + IVIG or
plasmapheresis
Treatment of thrombosis:
Current Recommendations
Erkan study
Aspirin for primary thrombosis prevention in the
antiphospholipid syndrome: double-blind, placebo-controlled
trial in asymptomatic antiphospholipid antibody-positive
individuals.
APLASA: multicenter, randomized, double-blind, placebo-
controlled clinical trial
Observational parallel study.
Conclusions:
aPL-positive individuals do not benefit from low-dose aspirin for
primary thrombosis prophylaxis
have low overall annual incidence rate of acute thrombosis and
develop vascular events when additional thrombosis risk factors
are present.
(Erkan D et al. Arthritis Rheum 2007; 56: 2382-2391).
Treatment of thrombosis in APS

Oral anticoagulation: problems
Bleeding
Frequent monitoring
Patient compliance with medication and diet
Prevention of thrombosis in APS:
Current problems


In patients with previous thrombotic event(s):
Oral anticoagulation at high INR vs. moderate INR ??
Most recommendations based on retrospective studies.
(Khamashta et al, NEJM, 1995; Krnic et al, Arch Intern Med, 1997
Prospetive studies: Crowther et al, NEJM, 2003; Finazzi et al, JTH,
2005)

Patients with aPL and no thrombosis (low dose aspirin vs. no treatment?)


Unresolved questions
Do patients with stroke require same level of
anticoagulation vs. those with DVT only?
Is aspirin or other anti-platelet agents alone,
sufficient?
Do we discontinue oral anticoagulation in
some patients when an additional risk factor
is no longer a problem (i.e. contraceptives)?

There is a need for more safer and
efficacious modalities of treatment for
thrombosis in APS.


Understanding the molecular and
intracellular events triggered by
antiphospholipid antibodies is important
in designing new modalities of targeted
therapies for treatment of APS.
Do aPL antibodies
cause/induce
thrombosis?
Pierangeli, S. S. et al. Circulation 1996;94:1746-1751
Exposed femoral vein and fiber-optic light
positioned under the vein in a mouse
Pierangeli, S. S. et al. Circulation 1996;94:1746-1751
Photograph taken from the video monitor demonstrating the
method for analysis of thrombus size
Thrombus formation
METHODS
aPL-IgG (500g) or
Control IgG
0 hr 48 hr 72 hr

Thrombus
Dynamics
Analysis
Antiphospholipid Antibodies
Promote Clot Formation in Mice
Un Visitante Ilustre
Micropoint laser to induce
thrombogenic injury.
How has the animal model of induced thrombosis
helped us in understanding aPL pathogenic effects?




Induction of thrombosis in a mouse model by IgG, IgM and IgA immunoglobulins from patients with the
Antiphospholipid Syndrome. Pierangeli et al. Thrombosis Haemost . 1995; 74: 1361-1367.

Generation and characterization of Monoclonal IgG Anticardiolipin Antibodies from a Patient with the
Antiphospholipid Syndrome. Olee et al. Proc Nat Acad Sci. (USA). 1996; 93: 8606-8611.
Identification of an Fc- receptor independent mechanism by which intravenous immunoglobulin (IVIG)
ameliorates antiphospholipid antibody-induced thrombogenic phenotype. Pierangeli et al. Arthritis
Rheum 2001;44: 876-883.
Arginine residues are important in determining the binding of human monoclonal antiphospholipid
antibodies to clinically relevant antigens. Giles et al. J Immunol 2006; 177: 1729-1736.
A human monoclonal anti-prothrombin antibody is thrombogenic.in vivo and upregulates expression of
tissue factor and E-selectin on endothelial cells. Vega-Ostertag et al. Br. J. Haematology. 2006;





APL antibodies and platelets


APL antibodies bind to platelet membranes. (Khamashta et al. Ann
Rheum Dis 1988; 47: 849-854).

Prothrombotic properties of antiphospholipid (aPL) antibodies may be
explained in part by their ability to enhance the activation of platelets
pre-treated with low doses of ADP, thrombin or collagen (Campbell
et al. Thromb Haemost 73: 519-524, 1995).

APL antibodies increase expression of GPIIb/IIIa and GPIIIa on
platelets pre-treated with low doses of a thrombin receptor agonist
peptide (TRAP) in a dose-dependent fashion. Hydroxychloroquine
reverses those effects in vitro (Espinola et al. Thromb Haemost, 2002;
87: 518-522).



Do APL antibodies affect platelet
activation in vivo?
Infusions of anti-GPIIb/IIIa (1B5) antibodies affect aPL-
mediated enhanced thrombus formation in a mouse model of
thrombosis.

aPL-antibodies do not enhance thrombosis in
3
-null mice.

Hydroxychloroquine diminishes platelet activation and
thrombus formation induced by aPL antibodies.
Edwards M et al. Circulation . 1997; 96:4380-4384.

Thrombogenicity of
2
glycoprotein I-dependent antiphospholipid
antibodies in a photochemically induced thrombosis model in
the hamster. Jankowski et al. Blood 2003; 101: 157-162.

Hydroxychloroquine in APS
Yoon KH. Sufficient evidence to consider hydroxychloroquine as an
adjunct therapy in antiphospholipid (Hughes) syndrome. J Rheumatol
2002; 29: 1222-1226.
Wallace DJ. Does hydroxychloroquine protect against clot formation in
systemic lupus erythematosus? Arthritis Rheum 1987; 30: 11435-1436.
Petri M. Hydroxychloroquine use in the Baltimore Lupus Cohort: effects
on lipids, glucose and thrombosis. Lupus 1996; 5: S16-22.
McCarty GA and Cason TE. Use of hydroxychloroquine in
antiphospholipid antibody syndrome at three academic rheumatology
units over two years: improvement in antibody titer and symptoms
management (abstract). 7th International Congress on SLE and Related
condictions. Abstracts Book, NY 2004.. pM17A.
Question
aPL antibodies enhance platelet activation in
vitro and in vivo.

What are the intracellular pathways
involved in aPL-mediated platelet
activation?
Intracellular events mediated by
aPL on platelets.
APL induce platelet activation and thromboxane
formation and platelet-derived thromboxane urinary
metabolites. (Martinuzzo ME et al. Thromb Haemost 1993; 70:667-
671 and Forastiero R et al. Thromb Haemost 1998; 79:42-45)
APL/anti-
2
GPI Abs induce production of
thromboxane A2 that is inhibited by cyclic-AMP
agonists. Indomethacine and phosphodiesterase
inhibitors such as theophylline inhibit TXA2. (Robbins
DL et al. J Rheumatol. 1998; 25: 51-56 and Opara E et al. 2003; 30: 55-
59).

p38- P
p38
ERK1/ERK2-P
F
(
a
b
)

2

I
g
G
N
H
S

F
(
a
b
)

2

a
P
L
1

F
(
a
b
)

2

a
P
L
2

F
(
a
b
)

2

a
P
L
3


F
(
a
b
)

2

a
P
L
4

F
(
a
b
)

2

a
P
L
5


P
B
S


P
B
S

+Thrombin 0.005 U/ml +Thrombin 1U/ml
43
43
43
Kda
A
B
C
Phosphorylation of p38MAPK and ERK1/ERK2 by aPL and thrombin
Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911-2919

Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911-2919
Vega-Ostertag M et al. Arthritis Rheum 2004; 50: 2911-2919






P38
cPLA
2

AA

PKC
Fibrinogen
gp IIIa
gpIIb
Ca
2+
PLC
PLC
DG
IP3
Ca
2+

TXB2
(+)
TX Rc

Dimerized

2
-glycoprotein I

Full activation
SB203580
Aspirin
Hydroxy-
chloroquine
(-)
(-)
Anti-GPIIbIIIa antibody
Receptor Antagonist
(-)
PS, PE
EXPOSITION
GPIb
(+)
Receptor recognized by aPL on
platelets
APO ER2 Lutters BC et al. J Biol Chem 2003; 2778: 33831-
33838.
Glycoprotein Ib/IX-V. Shi et al. Arthritis Rheum 2006; 54:
2558-2567.
Dimers of
2
GPI.


In previous studies, a chimeric
fusion protein was constructed by
the dimerization domain (apple 4) of

2
GPI .
As a control the monomeric protein
apple 2-
2
GPI which is not able to
form dimer - was constructed.
The authors demonstrated that
dimeric
2
GPI mimics in vitro the
effects of
2
GPI anti-
2
GPI
antibodies complexes. [Lutters, et al.
JBiol Chemistry 2001; 276:5 , 3060-3067]





Effects of B1 APOER2 on thrombus formation and platelet
aggregation in vivo
Treatment Thrombus size Platelet
aggregation
Dimer of
2
GPI 3629 562 m
2
56.3 % (n=3)
Monomer control 690 50 m
2
10% (n=3)
B1D APOER2 532 147 m
2

23% (n=3)




Antiphospholipid Antibodies
and Endothelial Cells
APL antibodies activate endothelial
cells in vitro and in vivo.
aPL or anti-
2
GPI antibodies upregulate EC adhesion
molecules and this effect is related to aPL binding to EC
(Del Papa N et al Arthritis Rheum 1997; 40: 551-561.
aPL-induced upregulation of ICAM-1, VCAM-1 and E-
selectin on HUVEC and increased adhesion of monocytes
to EC in the presence of
2
GPI (Simantov R et al J Clin Invest 1995;
96: 2211-2219).
Soluble levels of VCAM-1 significantly increased in plasma
of patients with APS and recurrent thrombosis (Kaplanski G et
al 2000; 43: 55-64).



1999;163:2922)
Activation of endothelial cells in vivo

Assessed by adhesion of leukocytes in
the microcirculation of the cremaster
muscle.
The number of leukocytes sticking within
five different venules is determined.
Adhesion is defined as leukocytes
remained stationary for at least 30
seconds.

Peter FW, et al. Microsurgery. 1998; 18:23-28.


APL antibodies enhance thrombus formation and this
correlates with activation of endothelial cells in vivo







Antiphospholipid antibodies from patients with Antiphospholipid Syndrome activate
endothelial cells in vitro and in vivo. Pierangeli et al Circulation, 1999; 99:1997-2002.
GDKV-induced antiphospholipid antibodies enhance thrombosis and activate
endothelial cells in vivo. Gharavi et al J Immunol 1999; 163: 2922-2927.
Functional analysis of patient-derived IgG monoclonal anticardiolipin antibodies using
in vivo thrombosis and in vivo microcirculation Models. Pierangeli et al. Thrombosis
Haemost 2000; 84:388-395.
Thrombogenic effects of antiphospholipid (aPL) antibodies are mediated by
intercellular cell adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1
(VCAM-1) and P-selectin. Pierangeli et al. Circ Res 2001; 88: 245-250.
E-selectin mediates pathogenic effects of antiphospholipid antibodies. Espinola et al
Thromb Haemost. 2003; 1:843-848.



aPL antibodies upregulate
tissue factor expression
Upregulation of tissue factor may account for
arterial and venous thrombosis.
Increased expression of TF on monocytes by aPL.
(Dobado-Barrios M et al Thromb Haemost 1999; 82: 1578-1582)
Inhibition of TF upregulation in monocytes by
dilazep (Zhou H et al, Blood 2004; 104: 2353-2358).
Increased sTF and VEGF in plasma of patients with
APS (Williams FM et al Thromb Haemost 2000; 84: 742-746; ,
Forastiero RR et al J Thromb Haemost 2003; 10:2250-2251; Cuadrado
MJ et al J Thromb Haemost 2006; 4:2461-2469)

Upregulation of TF by aPL on EC
Summary of our results

aPL increased TF expression on EC (1.8-4.2 fold increase)
The increase in TF expression was dependent on the dose of
antibody utilized.
TF function was increased by aPL (1.4 to 3.8 fold increase)
TF function increase was dependent on the dose of antibody
utilized.
TF expression was inhibited by MG132 (10-100%) and SB203580
(50-100%).
TF function was inhibited by SB203580 (34-54%).
aPL induce upregulation of IL-6 and IL-8.
Vega-Ostertag ME et al Arthritis Rheum 2005; 52: 1545-1554.

Effects of aPL on
phosphorylation of p38 MAPK
Effects of aPL on TF mRNA expression
(RT-PCR) in EC)
Conclusions


aPL induce phosphorylation of p38MAPK
aPL induce iNOS
aPL induce transcription of TF mRNA and
this effect is inhibited by SB 203580 in a
dose-dependent fashion.
(Vega Ostertag M, et al. Arthritis Rheum, 2005; 52: 5: 1545-1554)


Involvement of p38 MAPK in aPL-mediated
effects in platelets, EC and monocytes.
Publication Cell type
Vega-Ostertag et al. Arthritis Rheum
2004; 50:2911-2919
Platelets
Lutters BC et al. J Biol Chem 2003;
278: 33831-33838
Platelets
Vega-Ostertag ME et al. Arthritis
Rheum. 2005; 52: 1545-1554
Endothelial cells
Simoncini S et al. Int Immunol 2005;
17: 489-500.
Endothelial cells
Bohgaki M et al. Int Immunol 2004;
16: 1632-1641
Monocytes
Lpez-Pedrera C et al. Arthritis
Rheum. 2006; 54: 301-311.
Monocytes
Experimental Design II
Injection schedule
IgG- APS (500g) i.p.
0 hr 48 hr 72 hr

1. Thrombus dynamics analysis
2. Adhesion of WBC to EC in cremaster
3. TF activity in carotid artery homogenates
4. TF activity in mouse peritoneal macrophages
5. ACL activity
IgG- NHS(500g) i.p.
In some experiments, mice were
treated i.p. with 25 mg/Kg of SB
203580 or saline 30 min. before the
IgG injections
THROMBOSIS MODEL IN MICE
Effects of a p38 MAPK inhibitor on aPL-
induced thrombosis in vivo.
Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828-1834.
Effects of a p38 MAPK inhibitor on aPL-
mediated endothelial cell activation in vivo.
Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828-1834.
Determination of TF activity in mouse
peritoneal macrophages
Procedure done in the animals immediately after the surgical procedures and
after they were sacrificed. Peritoneal macrophages obtained after lavage of the
peritoneal cavity with 5 ml sterile PBS.
Two x 10
6
peritoneal cells were washed twice with PBS and resuspended in 1 ml
of Tris buffer saline-0.1% Triton X-100 pH 7.4 and centrifuged at 14,000 rpm
during 30 minutes. The cells were then washed twice and then resuspended in
50 l TBS-0.1% Triton X-100 and sonicated.
The TF activity of peritoneal cells lysates determined using a commercial
chromogenic assay (Actichrome TF, American Diagnostica, Stamford, CT) that
measures factor Xa after activation by the TF-Factor VII complex. The amount of
factor Xa generated is measured by its ability to cleave Spectrozyme Xa, a highly
specific chromogenic substrate for factor Xa.
Results expressed in pM/100 g tissue.
Effects of a p38 MAPK inhibitor on aPL-induced TF
activity in mouse peritoneal macrophages
Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828-1834.
Determination of TF activity in carotid artery homogenates.

Pieces of approximately 5 mm of uninjured carotid arteries were
dissected from both sides in each animal and were collected in a TBS-
0.1% TritonX-100 buffer containing heparin as anticoagulant.

The samples were homogenized. Homogenates of pooled carotid artery
from four animals in each group were washed once with the same buffer
and twice with TBS-0.1% TritonX-100. Finally the preparations were
resuspended in 50 mL of this buffer and sonicated.

The TF activity of lysates was determined using a commercial
chromogenic assay (Actichrome TF, American Diagnostica, Stamford,
CT).


Effects of a p38 MAPK inhibitor on aPL-induced TF
activity in carotid artery homogenates of mice
Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828-1834.
Platelet aggregation
Mouse blood was obtained in acid citrate dextrose anticoagulant (9/1
volume/volume) by cardiac puncture . Platelet rich plasma (PRP) was
obtained by centrifugation for 20 min at 120g.
Aggregation of platelet in PRP was measured turbidimetrically using a
dual channel aggregometer (Minigator II) following calibration with
platelet-poor plasma (PPP) at a stirring speed of 800 rpm. PRP was
adjusted to 240,00 platelets/ mL with PPP.
Aliquots (250mL) of PRP were placed in cuvettes containing magnetic stir
bars, warmed at 37
0
C and stirred for 1 min to obtain a stable baseline.
Aggregation was induced by addition of 0.005 U/mL of thrombin in light
transmission was recorded for 5 min.
Effects of a p38 MAPK inhibitor on aPL-induced
platelet aggregation
Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828-1834.
Experimental Design
Ex vivo experiments:
Determination of VCAM-1 in flat aorta preparations of mice using Qdot
conjugates and dual photon confocal microscopy.
Mouse arteries were pressure-perfused with 10% formalin. After fixation, the
arteries were washed three times with PBS.
The immunohistochemical procedure was done on a 24 well-plate. Blocking was
done with 2% BSA/5% goat serum for one hour. Primary antibodies were
incubated overnight at 4C. Washing steps were done to remove primary antibody
excess. Qdot-conjugated secondary antibody (Qdot Corp) was incubated for one
hour. Finally, nuclear visualization was done with Hoechst stain.
Image collection was done using a Zeiss LSM 510 Meta two-photon microscope
equipped with a near-infrared (NIR) titanium-sapphire femtosecond laser (Mira 900
Ti:S Coherent) tuned and mode-locked at 750 nm. The separation of the
emission signals was performed by acquisition of lambda stacks with posterior
selection of reference spectra using the META detector.
The following primary antibodies were used:, monoclonal rat anti-mouse VCAM-1
IgG (1:50 dilution, BD Pharmingen) and a nonimmune primary used to address the
contribution of nonspecific Fc receptor-mediated binding (nonimmune purified rat
IgG2a (BD Pharmingen). The following Qdot-bioconjugate was used in the
experiments: Qdot 655 goat F(ab')2 anti-rat IgG Conjugate

Effects of a p38 MAPK inhibitor on aPL-induced VCAM-1 expression in
aorta of mice ex vivo: nano crystals Q dot conjugates and dual photon
confocal microscopy.
Vega-Ostertag ME et al J Thromb Haemost 2007; 5: 1828-1834.
Conclusions
A p38 MAPK inhibitor (SB 203580):
Effectively diminished in vivo IgG-APS-
induced
thrombus formation
endothelial cell activation
platelet aggregation
tissue factor activity (carotid EC and
peritoneal macrophages)
VCAM-1 expression (aorta EC)
NF- B
NF-B is a complex group of heterodimeric and homodimeric
transcription factors that are trapped in the cytoplasm as an inactive
complex by I-B.
NF-B involved in transcription of inflammatory genes such as: IL-6, IL-8,
TNF- and IL-1 and in induction of adhesion molecules on EC (VCAM-1, E-sel
and ICAM-1) and in recruitment of inflammatory cells to extravascular sites.
NF-B associated with rheumatoid arthritis and other autoimmune
diseases.
APL antibodies and NF- B

Intracellular events in EC
induced by aPL antibodies:
aPL induce activation of
NF-B and correlates
with EC activation in
vitro and in vivo and
with thrombosis in vivo.
Espinola RG et al: J Thromb Haemost, 2003; 1:
843-848.
Dunoyer-Geindre S. et al. Thromb Haemost. 2002;
88: 851-857.
Bohgaki M, et al. Int Immunol. 2004; 16: 1632-1641.

MG 132
NF-B inhibitors used in RA and other autoimmune and inflammatory
diseases.
MG 132 = Carbobenzoxyl-leucinyl leucinylleucinal (Z-Leu-Leu-Leu-aldehyde; Z-
LLL-CHO).
MG 132 is a potent 20 S proteasome inhibitor that has been shown effective in
suppressing NF-B activation in different cellular systems.
Several studies have shown beneficial effects of MG132 on models of
rheumatoid arthritis, suggesting that this inhibitor may provide a new approach in
the treatment of this autoimmune disease.

Objectives
Are NF-B inhibitors effective in reversing
pro-inflammatory and pro-thrombotic effects
of aPL in vivo?

Experimental Design
IgG -APS (500g) i.p
+ 10 M MG 132 or DMSO:PBS (60:40).
0 hr 48 hr 72 hr

1. Thrombus dynamics analysis
2. Adhesion of WBC to EC in cremaster
3. TF activity in carotid artery homogenates
4. TF activity in mouse peritoneal macrophages
5. ACL activity
IgM - NHS (500g) i.p.
+ DMSO:PBS (60:40)
Inhibition of aPL-induced thrombus formation by
MG 132
Inhibition of aPL-induced EC
activation in vivo by MG 132
Effects of MG132 on TF activity on
mononuclear cells of mice treated with aPL
Antibodies
Effects of MG132 on TF activity on
homogenates of carotid artery of mice
treated with aPL Antibodies
Conclusions
IgG-APS enhanced thrombosis, endothelial
cell activation and tissue factor in vivo in
mice.
These effects were significantly diminished
by pre-treatment of the mice with MG132.
Montiel-Manzano et al. NY Acad Med 2007; 1108: 540-553.
Inhibitors of p38MAPK and NF-B for APS?
P38 MAPK and NF-B inhibitors effective in
diminishing in vitro and in vivo effects of aPL
Abs.
Can we use specific inhibitors of p38 MAPK
or NF-B to revert/ameliorate pathogenic
effects of aPL Abs?
Clinical trials are needed.

Antiphospholipid Antibodies
and the statins
Pleiotropic effects of statins
- TPA and PA inhibitior-1 expression
- Expression of adhesion molecules
- Pro-inflammatory cytokines
- Expression of tissue factor
- Thromboxane A2 synthesis and platelet reactivity
- Endothelin 1 synthesis
- NF-B activation
- MHC class II antigen expression

aPL antibodies and fluvastatin

Fluvastatin reduces aPL-induced adhesion of leukocytes and expression
of adhesion molecules on EC in vitro. (Meroni PL, et al. Arthritis and Rheum 2001;
44:2870-2878).

Fluvastatin abrogated thrombogenic effects of aPL antibodies in
vivo (Ferrara DE et al, Arthritis Rheum, 2003; 48: 3272-3279)

Fluvastatin inhibited the effects of the IgG-APS on tissue factor
expression. The effect was dependent on the dose of fluvastatin.
Mevalonate abrogated the inhibitory effects of fluvastatin on
expression of TF by aPL on endothelial cells. (Ferrara DE. et al, J Thromb
Haemost, 2004;2: 1558-1563).

Implications
These findings may have important
implications in designing new modalities
of treatment and prevention of recurrent
thrombosis in patients with APS.
Well designed clinical trials are needed to
investigate and confirm these findings in
APS patients
Objectives of the study
To determine the effects of statins on pro-
thrombotic and pro-inflammatory markers in
patients with aPL Abs
Patients
Four groups of 20 patients each
A) PAPS
B) Asymptomatic APS with persistently
positive aCL, LA and/or anti-
2
GPI tests.
C) SAPS
D) Asymptomatic SAPS with with persistently
positive aCL, LA and/or anti-
2
GPI
tests.
Inclusion/Exclusion criteria
aCL titers > 40 GPL or MPL units
Anti-
2
GPI > 99
th
percentile of normal controls.
>18 years of age.
No pregnant women.
Exclude if they are on statins or with pulse therapy with
steroids.
Not excluded if they are on HQ, aspirin, heparin, warfarin,
low dose prednisone (5-10 mg/day).
Exclude patients with liver problems.
Study Intervention
Fluvastatin 40 mg/day for 5 months.
Blood will be collected at the screening visit,
at one, three, six and seven months later.
At five months patients will be instructed to
stop fluvastatin.
Outcome measures
Determination of aCL, LA and anti-
2
GPI
titers.
Determination of PCA and TF mRNA in
PBMC.
Determination of VEGF, sTF, sVCAM-1, IL-6,
IL-8 and TNF-.
aPL, complement, endothelial
cell activation and thrombosis
Complement and aPL Abs.

A murine C inhibitor (crry) reverses aPL-mediated pregnancy loss,
thrombosis and endothelial cell activation in vivo. (Holers W et al. J Exp Med;
2002; 2:211-220)
Recent studies have shown that uncontrolled complement activation
leads to fetal death in aPL-antibody-treated mice. (Girardi G et al. J Clin
Invest. 2003; 112: 1644-1654)
Heparin seems to prevent obstetrical complications by aPL by blocking
activation of complement and not by preventing placental
thrombosis(Girardi G et al. Nature Medicine. 2004; 10: 1222-1226)
C3 and C5 Deficient mice are resistant to thrombosis and endothelial cell
activation induced by aPL antibodies (Pierangeli SS et al. Arthritis Rheum 2005;
52: 2120-2124).
Hypocomplementemia has been reported in patients with APS in three
studies. (Carbone J. et al. Lupus; 1999. 8:274-278; Munakata Y. Thromb Haemost.
2000; 83:728-731.Davis WD & Brey RL. 1992. Clin Exp Immunol 1992; 10:455-460).





Objective

Does an anti-C5 MoAb prevent aPL-
mediated thrombosis in mice?

Does an anti-C5 MoAb prevent aPL-mediated thrombosis in
mice?

Pierangeli SS et
al. Arthritis
Rheum 2005;
52: 2120-2124.
Effects of aPL Abs on thrombosis in C5aR deficient mice.
C5aR-/- + IgG-APS 3400 1681 108.9 33.4
C5aR-/- + IgG-NHS 777.3 270.4 0.8 0.4
C5aR +/+ + IgG-APS 3507 965 80.3 17.6
C5aR+/+ + IgG-NHS 1321 798 0.8 0.5
C5aR -/- + IgM-APS *676 690 96.4 30.8
C5aR-/- + IgM-NHS 958 388 0.0 0.0
C5aR +/+ + IgM-APS 3198 2361 99.8 31.4
C5aR+/+ + IgM-NHS 585 460 00.0 0.1
Romay-Penabad Z et al. NY Acad Sci 2007; 1108: 554-566.
We demonstrated that complement activation is a central
mechanism contributing to aPL antibody-induced
thrombophilia using three approaches:
a specific complement inhibitor (Crry-Ig)
genetically deficient mice (C3-/- and C5-/-)
Using specific anti-C5 Monoclonal antibodies.
Using C5aR deficient mice.


CONCLUSIONS
Further evidence of complement
involvement
Thrombus formation induced by antibodies to

2
glycoprotein I is complement dependent
and requires a priming factor (Fischetti et al Blood
2005; 106: 2340-2346).
Pathogenic aPL antibodies, in addition to their direct effects on platelet
and endothelial cell targets, induce complement activation, generating
complement split products which attract inflammatory cells that may
induce then thrombosis and tissue injury

Activation of complement may be a critical proximal
effector mechanism in aPL-associated thrombosis.
In APS patients, due to aPL IgG deposition targeted to the endothelium,
complement activation is increased locally and overwhelms normally
adequate inhibitory mechanisms.
Therefore, inhibition of complement activation should ameliorate
aPL-mediated vascular thrombosis.

IMPLICATIONS
APL antibodies in EC.
Receptor for aPL in EC?
Human
2
GPI binds to
endothelial cells through a cluster
of lysine residues that are critical
for anionic phospholipids binding
and offers epitopes for anti-
2
GPI
antibodies (Del Papa et al, 1997)
Treatment aCL titer in-vivo
(GPLU)
Thrombus size
(mm
2
)
APS-IgG + rDI 90.742.4 568.4325.0
APS-IgG + control 70.118.3 7959.02647

Five mice per group
DI/control infused at 40mg i.v 30 minutes before thrombosis
dynamics analysis


Effects of recombinant Domain I of

2
glycoprotein I on aPL-mediated thrombosis
TIFI and aPL Abs
TIFI is a a 20 amino acid
synthetic peptide (derived from
CMV) that shares similarity with
the PL- (membrane binding)
region of
2
GPI .
TIFI reduced aPL-mediated
thrombosis and EC activation
in vivo.
TIFI reduced the binding of
FITC-
2
GPI to target cells (EC,
and monocytes).
(Vega-Ostertag et al. Lupus 2006;
597: 247-256)
TLR-4: Receptor for aPL on EC?


MyD88 signaling cascade - associated to TLR-4 - is
triggered by aPL reacting with
2
GPI on the endothelial cell
surface membrane (Raschi et al. Blood 2003; 101: 3495-3500).
APL Abs are not thrombogenic in LPS -/- mice and EC
activation and TF are diminished. (Pierangeli SS et al Ann Rheum Dis.
2007; epub ahead of press)


Effects of various inhibitors on TF
upregulation by
2
GPI dimer.
Effects of various inhibitors on ICAM-1 expression induced by
dimers of
2
GPI
Conclusions
Animal models of thrombosis and endothelial cell activation
in APS have been instrumental in
Elucidating the pathogenic mechanisms induced by aPL
antibodies
Determining specific targets recognized by aPL antibodies.
Examining possible ways by which aPL antibodies are
induced.
Test new targeted therapies to prevent aPL-effects in
vivo.
WE NEED HUMAN STUDIES!
Animal models in APS
New Approaches to Prevention of Thrombosis in APS?


Statins: Fluvastatin reversed EC activation and TF upregulation by aPL antibodies in
vitro and abrogated enhanced thrombus formation and EC in vivo. In mice
Hydroxychloroquine: Decreased platelet activation induced by aPL antibodies in
vitro and inhibited aPL-mediated thrombosis in mice in vivo.
Antiplatelet agents: GPIIbIIIa inhibitors decreased aPL-mediated enhancement of
platelet activation and abrogated aPL-induced thrombus formation in mice.
p38MAPK inhibitors: In vitro effects on aPL-induced TF upregulation in EC. Effects
on aPL-mediated thrombus formation, EC activation, TF upregulation and platelet
activation in vivo.
NF-B inhibitors: In vitro effects on aPL-mediated upregulation of TF. Significant
decrease in some aPL-enhanced thrombosis, TF upregulation,
Specific complement inhibitors: anti-C5 Monoclonal antibody decreased aPL-
mediated thrombus formation.
Specific inhibitors/blocking agents to the receptor(s) in target cells: TLR-4
inhibitors? Peptides that mimic regions of
2
GPI?



New Approaches to Prevention of Thrombosis in APS?

ACE inhibitors: Inhibit monocyte TF expression

Dilazep, dipyridamole: Adenosine uptake inhibitor; antiplatelet;
inhibits monocyte TF expression.

LJP 1082:
2
GPI-specific B cell toleragen; decreases anti-
2
GPI-
specific B cell toleragen;decreases anti-
2
GPI antibody levels.

Rituximab: Anti CD20.



New Approaches to Prevention of Thrombosis in APS?

ACE inhibitors: Inhibit monocyte TF expression

Dilazep, dipyridamole: Adenosine uptake inhibitor; antiplatelet;
inhibits monocyte TF expression.

LJP 1082:
2
GPI-specific B cell toleragen; decreases anti-
2
GPI-
specific B cell toleragen;decreases anti-
2
GPI antibody levels.

Rituximab: Anti CD20.



Collaborators

Mariano Vega-Ostertag, MS Morehouse School of Medicine
Zurina Romay-Penabad, PhD UTMB
Guadalupe Montiel, B.S. UTMB
Elizabeth Papalardo, B.S. UTMB
Dardo E. Ferrara, MD Morehouse School of Medicine
R.G. Espinola, MD Morehouse School of Medicine
X. Liu, MD Morehouse School of Medicine
Ian P. Giles University College London
Robert Swerlick, MD Emory University School of Medicine
Pier Luigi Meroni, MD University of Milan
Guillermina Girardi, PhD Hosp Spec Surgery
Jane Salmon, MD Hosp Spec Surgery
VM Holers, MD Univ Colorado,Denver
Philip deGroot Utrecht University.




The 13
th
International Congress on antiphospholipid antibodies:
Galveston, TX. Spring 2010.
Acknowledgements
These studies were partially funded by a
Research Centers in Minority Institutions -
National Institutes of Health grant # G12-
RR03034 and a Minority Biomedical
Research Support Grant from the National
Institutes of Health (GM58268-02) and a
multidisciplinary clinical research grant NIH
grant #: 2P60AR047785-06.