Dr. Ertan yetkin

1
Molecular Mechanism of Aortic Stenosis Loutraki,
Athene,

• Dr. Ertan yetkin
• Abant Izzet Baysal University Faculty of Medicine
  Department of Cardiology, Bolu Turkiye

Prof. Dr. Ertan Yetkin
2
Headings

• Normal Aortic Valve
• Calcific Aortic Valve
• Aortic Stenosis Atherosclerosis
• Mechanism
• Calcification and Bone
• Angiogenesis
• Extracellular Matrix Remodeling
• Infection Inflammation
• Treatment
• Future Perspectives

3

• Calcific aortic stenosis (AS) is the most common
  cause of aortic valve replacement in developed
  countries, and this condition increases in
  prevalence with advancing age, afflicting 23 of
  the population by the age of 65 years.

4

• Prolonged wear and tear and age associated
• On the other hand rheumatic etiology is the still
  most common cause of aortic stenosis in
  developing or underdeveloped countries.

5
Normal Aortic Valve

• Smooth
• Thin
• Opalescent
• Clearly defined tissue layers

6
Normal Aortic Valve

• Histologically, the aortic valve consists of
  three principal layers, the ventricularis at the
  inflow surface containing collagen and elastin,
• The spongiosa in the center composed of
  glycosaminoglycans
• The fibrosa the outflow surface containing
  densely packed collagen fibers.
• The outer surface of the valvular leaflets is
  lined by endothelial cells that cover
  interstitial mesenchymal cells located throughout
  the leaflets.

7

• Normally, aortic valves are avascular and their
  oxygen supply is provided by diffusion from the
  blood stream.
• Until recently, the concept was generally
  accepted that calcific aortic valve disease is a
  degenerative and unmodifiable process, basically
  induced by long-lasting mechanical stress.
• Although calcification and ossification in aortic
  valves has been described, little is known about
  the synthesis of bone matrix proteins in calcific
  aortic valve stenosis.

8
Histology. Hematoxylin and eosin staining.
Representative low-power photomicrographs of
control valve (Panel A) and stenotic valve (Panel
B). The control valve is thin and shows a
preserved three-layer structure. The stenotic
valve shows marked thickening, fibrous disarray,
and focal calcification (asterisks).
Kaden JJ et al, Atherosclerosis 2003
9
Histopathology

• The pathologic changes associated with early AS
  include
• valve thickening,
• accumulation of irregular fibrocalcific masses,
• basement membrane disruption,
• collagen fiber disarray,
• inflammatory infiltrate consisting predominantly
  of macrophages and lymphocytes.
• The fibrotic thickening and calcification are
  common eventual endpoint in both nonrheumatic
  calcific and rheumatic aortic stenosis.

Otto CM et al Circulation 1994
10
Valvular Calcification

• is an active process
• cardiovascular calcification is composed of
  hydroxyapatite deposited on a bone-like matrix of
  collagen, osteopontin, and other minor bone
  matrix proteins.
• Osteopontin expression has been demonstrated in
  the mineralisation zones of heavily calcified
  aortic valves obtained at necropsy and surgery.

11
In situ hybridization localizes osteopontin mRNA
to a subset of lesion macrophages. Hybridization
with a 35S-labeled antisense riboprobe
demonstrates the presence of osteopontin mRNA (A,
black autoradiography grains, left) in cells in
an aortic valve lesion, whereas hybridization
with the sense (control) riboprobe demonstrates
no specific hybridization (B). The anti-CD68
antibody identifies these cells as macrophages
(C, black immunoreaction product, left),
OBrien KD, Circulation 1995
12

• Surgically exiced valves have shown areas of
  mature lamellar bone, haemopoetic marrow, and
  bone remodelling.,

Mohler ER, et al Circulation 2001
13
Medium-power photomicrograph of aortic valve
showing BMP-2/4 staining of lamellar bone with
predominance of staining in cytoplasm. BMP-2/4
was expressed by myofibroblasts and
preosteoblasts in areas adjacent to B-cell and
T-cell lymphocytic infiltration. Hematoxylin and
eosin, original magnification x450
14
Bone Regulatory Proteins

• Bone morphogenetic proteins 2 and 4 (BMP 2/4),
  potent osteogenic morphogens, were shown to be
  expressed by myofibroblasts and preosteoblasts in
  areas of the valves, where ossification was
  identified.

15

• After stimulation with TLR2 and TLR4 agonists,
  aortic valve interstitial cells express higher
  levels of the pro-inflammatory and pro-osteogenic
  mediators BMP-2, and greater osteogenic
  phenotypic changes (alkaline phosphatase
  activity, calcified nodule formation) than
  pulmonary valve interstitial cells.

16
 Stimulation With PGN and LPS Induces Calcified
Nodule Formation in AVICsAVICs and PVICs were
cultured in conditioning medium (CM) and
incubated with PGN or LPS for 28 days. Alizarin
red S stains (representative of 3 experiments)
show more and bigger calcified clusters in AVICs
than PVICs. The upper right inset shows an
Alizarin red S-stained stenotic aortic valve as a
positive control. Arrows indicate calcium
deposits
Tang et al JACC 2008
17
Receptor activator of nuclear factor ?B Ligand
(RANKL) and osteoprotegerin (OPG)

• Increased expression of osteocalcin and cbfa1
  have been demonstrated in calcified human aortic
  valves indicating localized osteoblastic
  differentiation.
• Receptor activator of nuclear factor ?B Ligand
  (RANKL) and osteoprotegerin (OPG) are members of
  a cytokine system involved in bone turnover and
  vascular calcification.

18

• In cultured human aortic valve myofibroblasts,
  RANKL promotes matrix calcification and induces
  the expression of osteoblast-associated genes,
  indicating a transition towards an osteogenic
  phenotype.

19

• RANKL and OPG are differentially expressed in
  calcific aortic stenosis. While marked expression
  of RANKL is noted in calcified valves, OPG is
  strongly expressed in normal aortic valves. Mice
  deficient for OPG, have developed vascular
  calcification and shown an expression of RANKL in
  calcified areas.
• These observations provide clear molecular
  mechanisms of localized tissue calcification
  including the calcification of valvular
  structures.

20
Fig. 1. Western blot and immunohistochemistry for
OPG. (A) Western blot of native valve protein
extracts for OPG showing distinct bands at 40 kDa
in control valves (n  3) and no bands in
stenotic valves (n  5) (top). Coomassie staining
of gels demonstrates equal loading of wells
(bottom). (B) OPG immunostaining of a control
valve showing low cellularity and a high
proportion of OPG-positive cells. (C) OPG
immunostaining of a thickened, but not calcified
area of a stenotic valve. Cellularity is
increased but the proportion of OPG-positive
cells (arrows) is decreased. (D) OPG
immunostaining of a calcified region from the
same stenotic valve as in (C), showing scattered
cells weakly positive for OPG (arrows) and
unspecific staining of focal calcification
(arrowheads). (E) Immunostaining with an
isotype-matched IgG control antibody showing
intense staining of focal calcium deposit
(arrowheads) and absent staining of non-calcified
tissue. (F) Control immunostaining of a
thickened, but not calcified area of the same
stenotic valve as in (E), showing no staining.
Original magnification 200.
Kaden JJ, et al J Mol Cell Cardiol 2004
21
Fig. 3. Immunohistochemistry and western blot for
RANKL. (A) RANKL immunostaining of a stenotic
valve demonstrating positive cells in the
subendothelial space. (B) RANKL immunostaining of
a stenotic valve demonstrating positive cells and
a focal calcium deposit. Unspecific staining of
calcium (arrowheads). (C) RANKL immunostaining of
a stenotic valve demonstrating positive cells
(arrows) in association with focal calcium
deposit. Unspecific staining of calcium
(arrowheads). (D) RANKL immunostaining of a
control valve showing scattered-positive cells.
(E) Western blot of native valve protein extracts
for RANKL showing bands at 45 kDa in stenotic
valves (n  3) and no bands in control valves
(n  3) (top). Coomassie staining of gels
demonstrates equal loading of wells (bottom).
Original magnification 200 except for panel B,
original magnification 100.
22
Bone mineral density and aortic stenosis

• Elevated serum calcium and phosphate levels and a
  high calcium-phosphate product and parathormone
  level (from secondary hyperparathyroidism) have
  been almost invariably documented, in patients
  with valvular calcification undergoing dialysis.
• Accordingly, these abnormalities of calcium and
  phosphate metabolism and their duration are
  likely to be important factors predisposing to
  calcific aortic stenosis and its rapid
  progression in long-term uremia.
• Additionally, bone mineral density was found to
  be significant, but inversely associated with
  aortic valve calcification and mitral annular
  calcification as well, although the causative
  relationship between bone mineral density and
  valvular calcification is not documented yet.

Aksoy Y, et al Coron Artery Dis 2007
23
Non-rheumatic Aortic Stenosis and Atherosclerosis

• Histopathologic evidence suggests that early
  lesions in aortic valves are not just a disease
  process secondary to aging, but an active
  cellular process that follows the classical
  response to injury hypothesis similar to the
  situation in atherosclerosis.
• Pathological studies of stenotic aortic valves
  have suggested that degenerative, non-rheumatic
  valvular AS has some similarities to
  atherosclerotic lesions.

24
Hypercholesterolemia

• The growing body of literature suggests that
  hypercholesterolaemia may play a role in aortic
  valve calcification.

25

• By confocal microscopy, Sarig and colleagues
  found that cholesterol is contained in the centre
  of calcified granules of coronary artery
  atherosclerotic plaques, which suggests that
  lipids are involved in the precipitation of
  calcium and mineral crystals.

Sarig S, et al Lab Invest 1994
26
Risk factor for aortic valve calcification

• Age
• Male gender
• Hypertension
• Diabetes mellitus
• Hypercholesterolemia
• Smoking
• Hyperparathyroidism
• Renal disease
• Decreased bone mineral density

27

• Microscopically, there was increased leaflet
  thickness, lipid accumulation, collagen disarray,
  and calcific deposits.
• Early lesion in AVS and Atherosclerosis
• Basement membrane disruption
• Lipid accumulation
• Accumulation of macrophages, and T cells
• Otto CM, Circulation 1994

28

• Weiss et al have recently shown that
  hypercholesterolemic LDLr/ApoB100/100 mice are
  prone to develop calcification and oxidative
  stress in the aortic valve, with functional
  valvular heart disease, mimicking the clinical
  syndrome.
• Weiss RM, et al Circulation 2006

29
Von Kossa staining of aortic valve tissue.
Mineralization of tissue appears as discreet dark
foci. L indicates leaflet LA, leaflet
attachment. Bar left0.3 mm
30

• Additionally, angiotensin-converting enzyme (ACE)
  and angiotensin II type 1 and type 2 receptors
  are present in stenotic aortic valves,
  implicating this signaling pathway in the disease
  process.

31
ACE in a human aortic valve lesion. A, Double
immunostaining for macrophages (blue stain) and
ACE (red stain) demonstrate that the majority of
macrophages are blue, indicating the absence of
ACE protein. A minority of macrophages contain
ACE protein, identified by their purple stain.
In contrast, the vast majority of red ACE
staining is extracellular. B, Double
immunostaining for macrophages (blue stain) and
apoB, the primary protein of LDL cholesterol
particles (brown stain), demonstrate the
presence of extensive extracellular apoB
staining, which colocalizes with extracellular
ACE. Original magnification x400.
OBrien KD, Circulation Arch Int Med 2005
32
Colocalization of apoB, ACE, and angiotensin II
(AngII) in a human aortic valve lesion.
Single-label immunostaining (black stains) was
performed on adjacent sections using antibodies
to apoB (A), ACE (B), and AngII (C). ApoB and ACE
are colocalized. AngII, the enzymatic product of
ACE, also colocalizes in the regions that contain
ACE. Original magnification x40, methyl green
counterstain.
33
(No Transcript)
34
Angiogenesis and Growth Factors

• Angiogenesis is defined as the outgrowth of new
  capillaries from pre-existing capillaries and is
  regulated by a balance between angiogenic
  activators such as VEGF, FGF-2 and PDGF, and
  angiogenic inhibitors.
• It has been reported that the activation of
  angiogenesis in aortic valves occurs in a close
  association with valvular stenosis, particularly
  with calcified aortic valve stenosis.
• Calachour et al have demonstrated that the
  formation of angiogenic sprouts from stenotic
  valves occurs significantly faster than from
  non-stenotic valves.

35
(AN) A non-stenotic aortic valve is stained with
HE (A). CD31 staining (B) and immunostaining for
vWF (C) are visible in endothelial cells (arrows)
covering the cusps of the aortic valve. A
stenotic aortic valve is stained with HE (D).
Immunostaining for CD31 (E) and for vWF (F)
reveals an intense vascularization (arrow) of the
stenotic aortic valve. Several small immature
blood vessels (small arrows) are stained only for
CD31. CD34 immunostaining is not only visible in
endothelial cells (arrows) covering the cusps but
also in some single cells (arrowheads) within the
tissue of the non-stenotic valve (G, see also the
inset in higher magnification), whereas
immunostaining for Tie-2 is present only in
endothelial cells (arrows) covering the cusp of
the non-stenotic valve (H). In contrast, CD34 (I)
and Tie-2 (J) immunostaining is not only present
in endothelial cells covering the cusps but also
in numerous blood vessels (arrows) and single
cells (arrowheads) within the valvular tissue of
the stenotic aortic valve. There is no staining
for CEACAM1 in the non-stenotic aortic valve (K)
whereas small vessels (arrow) and some single
cells (arrowheads) within the tissue of the
stenotic aortic valve exhibit CEACAM1 staining
(L). All sections are counterstained with Calcium
red to visualize the tissue structure and enable
a better assessment of the localization of the
specific immunostaining (dark brown staining).
The magnification for all panels AI and K is
350 and for the panels J and L it is 700. The
magnification of the inset in panel G is also
700.
Chalajour F, Exp Cell Res 2004
36
Tissue explants from stenotic and non-stenotic
aortic valves in AVA. Capillary-like sprouts from
the stenotic aortic valve at day 10 (A) and day
21 (B) observed with a phase contrast microscope.
Note the development of an intense network
between capillary sprouts (the marked area) after
21 days of culture in comparison to the same area
in A. In contrast, only a few capillary-like
sprouts are visible in AVA using tissue explants
from the non-stenotic aortic valve 28 days of
culture (C). Higher magnification of C reveals a
few sprouts with rare anastomoses (D). The
magnification for AD is 200.
37

• Immunohistochemistry for the endothelial cell
  markers CD31, CD34, von-Willebrand factor and
  Tie-2 and for the cell adhesion molecule reveals
  an intense vascularization of stenotic cusps in
  contrast to non-stenotic cusps.
• Correspondingly, it has been shown that
  endothelial cells of stenotic but not those of
  non-stenotic valves exhibit CEACAM1, a cell
  adhesion molecule expressed in angiogenic, but
  not in quiescent endothelial cells and involved
  in angiogenesis.

38

• Among the angiogenic factors,Vascular endothelial
  growth factor-A (VEGF-A)
• induce migration of EC
• proliferation of endothelial cells (ECs),
• enhance vascular permeability,
• modulate thrombogenicity
• is a potent inducer of the expression of
  matrix-degrading metalloproteinases

39
                                                
                              (A)
Immunostaining of the valvular structures with
FVIII-related antigen. Positive immunostaining
for the antigen can be seen in the walls of the
capillary structures. (B) Immunostaining with the
antibody to VEGF. Endothelial cells of the
capillary structures and stromal cells stain
positively. (C) Immunostaining with the antibody
to Flk-1. Immunostaining is similar to VEGF
showing positivity in endothelial cells and
fusiform stromal cells. (D) With Flt-1, the
distribution of immunostaining is similar to VEGF
and Flk-1. A different chromogen is used
(diaminobenzidine) showing the positive reaction
as a brown color. (E) With eNOS, strong staining
of capillary endothelial cells can be seen. (F)
With iNOS macrophages in the stromal tissue stain
positively. The endothelial cells appear negative
in this field.
40
TGF-ß

• Another potentially involved peptide in the
  pathogenesis of aortic stenosis is tranforming
  growth factor-ß (TGF-ß). Most of the effects of
  TGF-ß in the cardiovascular system known to date
  have been gathered for TGF-ß1. It stimulates the
  formation and deposition of extracellular matrix.
  
• TGF-ß1 has been shown in the nonrheumatic
  calcific aortic valve tissue.

41

• Pathologic angiogenesis is a hallmark of various
  proliferative diseases including tumor
  angiogenesis and atherosclerosis. Angiogenesis is
  also essential for longitudinal bone growth, and
  angiogenic factors such as VEGF are required for
  endochondral bone formation and fracture healing.
  
• The presence of neoangiogenesis in all ossified
  valves is consistent with the hypothesis that
  angiogenesis facilitates endochondral bone
  formation in calcified cardiac valves.
• Angiogenesis may be a key step in remodeling of
  the aortic valve and eventual formation of
  calcified valves.

42

• However, considering the differences in the time
  course of neo-angiogenesis, which takes several
  days while valve calcification lasts for years,
  angiogenesis is likely to be an epiphenomenon
  related to pathological inflammation.
• In terms of therapeuthic approaches, e.g blocking
  angiogenesis, it is difficult to identify a
  proper time window for an antiangiogenic approach
  to result in an efficient modification of the
  natural disease process.

43

• VEGF has been shown to play an important role in
  bone formation besides its angiogenic activity.
  Indeed, it has been shown that VEGF is
  upregulated at the sites of bone formation and is
  involved in bone healing.
• These data indicate that VEGF as an
  inflammatory mediator - plays a functional role
  in bone formation and thereby calcification.
• An anti-angiogenic treatment strategy (such as
  the use of Avastin) may not be a first line
  option in preventing aortic valve calcification.
• So it is reasonable to focus on ECM remodelling,
  inflammation and osteoblast-like differentiation
  rather than angiogenesis to prevent the
  progression of valve calcificaition.

44
Extracellular Matrix Remodelling in Aortic
Stenosis

• A number of ECM proteins normally found in bone,
  including
• osteocalcin,
• osteopontin,
• osteonectin,
• bone morphogenetic protein,
• matrix metalloproteinases- (MMP1,2,3,9 TIMP1,
  TIMP2),
• MMP-9, are present in cardiovascular
  calcifications, including calcified valves, but
  in general are not found in normal cardiovascular
  tissue. Matrix metalloproteinases are a group of
  zinc-dependent endopeptidases (collagenases,
  gelatinases and stromelysins).
• ALSO EXIST IN CALCIFIC AORTIC VALVES

45
                                                
                 Immunohistochemistry.
Representative photomicrographs of control (left)
and stenotic valve sections (right). Positive
immunoreactivity is shown by red staining, nuclei
are counterstained blue. (Panels A and B),
immunostaining for leukocytes (CD45). Marked
leukocyte infiltration occurs in the stenotic
valve. Only scattered leukocytes are demonstrated
in the control valve in association with an area
of mild subendothelial thickening. (Panels C and
D), immunostaining for IL-1ß. Marked staining in
a similar localization as the leukocyte
infiltrate. Only faint staining of scattered
cells is found in the control valve. (Panel E and
F), immunostaining for MMP-1. Intense
immunoreactivity in the stenotic valve in a
similar localization as IL-1ß. Positive cells
exhibit an elongated to stellate morphology
indicating activation. Weak staining of round
cells in the control valve. (Panels G and H),
negative control sections replacing pre-immune
mouse serum for primary antibodies.
Kaden JJ, Atherosclerosis 2003
46
Tenascin C

• Extracellular protein, Tenascin C has been shown
  in calcific aortic valve in association with
  MMP-2. Human calcific aortic cusps have
  demonstrated immunohistochemically prominent
  deposition of tenascin C, MMP-2.
• In aortic valve intertitial cell culture,
  tenascin C stimulated both MMP2 mRNA expression
  and MMP-2 gelatinolytic activity.
• Tenascin-C is a modular and multifunctional
  hexameric extracellular matrix (ECM) glycoprotein
  implicated in cell proliferation, migration,
  differentiation and apoptosis.
• Tenascin-C expression is less abundant in normal
  adult tissues, but is induced in malignant tumors
  and during inflammation and tissue repair.
• Tenascin-C expression has also been increasingly
  documented in vascular diseases, and very recent
  evidence suggests that there may be a link
  between protein expression and the development of
  calcific aortic stenosis.

47
                                                
     Figure 1. (A) A decalcified histologic
section of an aortic valve stained with
hematoxylin-eosin. Eosin-stained dystrophic bone
is seen between the endothelial lining of the
valve and the calcium nodule in the lower part of
the image. A cement line joins the ossification
to the calcium nodule. Dystrophic bone is marked
with an arrow. Scale BAR 100 µm. (B) Intense
tenascin-C (TN-C) immunostaining (arrows) can be
seen in the vicinity of the calcified areas
(marked with an asterisk) of the stenotic valve.
Notice the inflammatory cells and the
neovascularization (arrowheads) in the right
corner of the field. (C) TN-C immunoreactivity
can be seen as a basement membrane-associated
zone in undiseased valves (arrow). The valvular
stroma is otherwise negative. (D) Around the
areas of calcification (asterisk) in a stenotic
valve, intense TN-C immunopositivity (arrows) can
be seen. Linear basement membrane-associated
staining is missing. (E) Immunostaining with an
antibody to alpha-smooth muscle actin. Strong
positivity for this antigen can be seen in
stromal fibroblast-type cells, suggesting
myofibroblastic differentiation of the cells. (F)
In situ hybridization of a stenotic valve. Strong
signals for tenascin (arrows) can be seen in
fusiform fibroblast-like cells in the stroma.
Satta J et al J Am Coll Cardiol 2002
48
Tenascin-C expression presented in percentages
in relation to the degree of valve pathology.
Satta J et al, J Am Coll Cardiol 2002
49

• It has recently been shown that expression of
  cystatin C, a member of cystein proteases
  inhibitor is increased in calcific human aortic
  valve compared to normal valves.
• Additionally the expression pattern of cystatin C
  is quite similar to those of TGF-ß1 indicating
  an association between tgfb-1 and cystatin C in
  calcific aortic valve.
• It has been shown that TGF- ß 1 clearly regulates
  cystatin C expression in murine embryo, vascular
  smooth muscle.

Yetkin E, J Vasc Res under review 2009
50
TGF-b1 and Cyatatin C staining in calcified (A,
primary magnification x100) and normal (B,
primary magnification x200) human aortic valves
using immunohistochemistry.
51
Figure-III

• Similar expression pattern of Cys C and TGFb-1 in
  calcified aortic valve tissue. Immunohistochemical
  detection of Cys C in the calcifying (A1),
  endothelial and subendothelial area (A2).
  Immunohistochemical detection of TGFb-1 in
  calcifying (B1), endothelial and subendothelial
  area (B2) (Primary magnification x100).

52

• Expression of Cystatin C (A) and TGFb-1 (B) in
  capillarized and inflammatory areas of the
  diseased valves at a higher magnification
  (Primary magnification, x200)
• Nuclear and perinuclear staining of Cystatin C
  (A) and TGFb-1 (B) in chondroid tissue of the
  diseased valves (Primary magnification, x400)

53
Cyc CC
TGF beta-1
54

• In accordance with these findings Helske et al
  demonstrated increased expression of cathepsin
  enzymes S, K, and V and their inhibitor Cystatin
  C as well in human calcific aortic valves.

Heslske E et al Circulation 2007
55
Infection, Inflammation and Aortic Stenosis

• C.pneumonaie specific antigen or DNA has been
  shown in stenotic aortic valves and as well as in
  early degenerative aortic valve stenosis.
• With the use of immunohistochemical staining,
  polymerase chain reaction, and electron
  microscopy, C. pneumoniae has been detected with
  high frequency in mildly diseased and severely
  stenotic aortic valves and a high frequency of C.
  pneumoniae seropositivity, presumably reflecting
  chronic C. pneumoniae infection, has also been
  described in patients with aortic valve stenosis.
  
• However several studies have not demonstrated an
  association between C.pneumoniae and calcific
  aortic stenosis.
• Another infectious mechanism has been
  demonstrated in an animal model study. Cohen et
  al. has suggested that recurrent low grade
  endocarditis from calcifying oral bacteria,
  particularly when occurring with synergistic
  strains, may be one cause of calcific aortic
  stenosis.

56
Electron micrograph of stenotic aortic valve
showing Chlamydia-like particles. CM
cytoplasmic membrane OM outer membrane PS
periplasmic space. Bar 0.2 µm (35 reduction).
57
Immunostaining of a stenotic aortic valve with
Chlamydia speciesspecific monoclonal antibody,
showing a positive reaction (A). There is no
staining in normal aorta (B).
58

• In support of the inflammatory nature of calcific
  aortic valve disease, an association between
  systemic levels of hs-CRP and severe nonrheumatic
  calcific aortic stenosis has been described in
  selected patients free of coronary, carotid, or
  peripheral atherosclerosis.
• The association between hs-CRP and calcific
  aortic valve disease was independent of other
  risk factors for atherosclerosis, including BMI,
  but hs-CRP levels were not related to the
  severity of valve stenosis or to the degree of
  valve calcification.
• Increased expression of adhesion molecules namely
  intercellular adhesion molecule-1, vascular cell
  adhesion molecule-1, and E- selectin have been
  shown in degenerative aortic valves indicating an
  inflammatory process in calcific nonrheumatic
  aortic stenosis.

59
Inflamatory Markers

• hsCRP
• IL
• ICAM-1
• VCAM-1
• CD 45
• E-selectin
• C. pneumoniae

60
TREATMENT of AORTIC STENOSIS
61
Lipid Lowering and Progression of Aortic Valve
Stenosis

• The effects of statin treatment on aortic
  stenosis has gained importance recently based on
  the data of several trials.
• In one of these trials, the use of a statin was
  inversely associated with the progression of
  aortic stenosis.
• Regarding statins and their association with AS,
  there is evidence that statin therapy is
  associated with markedly lower hemodynamic
  progression of AS.
• Furthermore, Rosenhek et al have demonstrated
  that this effect is independent of cholesterol
  levels.
• Significant improvements in mean and peak aortic
  valve gradients, as well as serum lipid levels,
  were observed in the statin group of the RAAVE
  study (rosuvastatin affecting aortic valve
  endothelium), but not in the control group.

62
SALTIRE

• On the contrary, two recent randomized double
  blind studies revealed that statin treatment did
  not show any effect on the progression of aortic
  valve stenosis. One of them is the SALTIRE study,
  where intensive lipid-lowering therapy does not
  halt the progression of calcific aortic stenosis
  nor induced its regression.

63
SEAS

• Likewise, the simvastatin and ezetimibe in aortic
  stenosis study (SEAS) showed comparable aortic
  valve replacement rates in the simvastatin-ezetimi
  be treated group and in the non-treated group.
• Negative results of randomized double blind
  studies would potentially redirect the interest
  to other treatment modalities to retard the
  progression of aortic stenosis.
• It is possible that these studies may not rule
  out a small reduction in disease progression or
  clinical endpoints.

64
Selected clinical studies of statin therapy in
adults with aortic stenosis.
Statin No Statin p
Retrospective Novaro 2001 174 Shavelle 2002 Bellamy 2002 Rosenhek 2004 AVA 1.2cm2 AVA 1.32 cm2 Vmax 3.96 m/s AVA AVC AVA Vmax -0.06cm2 AVC12 per year AVA -3 per year Vmax 0.10 m/s per year -0.11 cm2 AVC32 per year AVA -7 per year Vmax 0.39 m/s pear year 0.03 0.006 0.04 0.0001
Prospective ONR RAAVE Trial 2007 Vmax 3.63 m/s Vmax Vmax -0.04 m/s per year Vmax _0.24 m/s per year 0.007
Prospective RC SALTIRE Trial 2005 SEAS Trial Vmax 3.43 m/s Vmax 3.2 m/s Vmax Major CV events Vmax 0.199 m/s per year Hazard ratio (HR) 0.93, 95 confidence interval (CI) 0.83_1.12 Vmax 0.203 m/s per year -- NS NS
ASTRONOMER Trial Vmax 3.1 m/s 1 DP AVA 2nd time to AVR or cardiac death aAgatston method.
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• One aspect became obvious that the calcifications
  as they appear in the atherosclerotic process are
  distinct from the calcification process in a
  degenerating aortic valve.
• This may already be one explanation for why
  statins demonstrated to be inefficient in
  preventing the progression of aortic valve
  stenosis, while they are associated with
  regression of atherosclerotic plaques.

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• Additionally, while reducing blood cholesterol
  levels, statins might have induced bone
  formation.
• A recent animal study showed that statins induce
  BMP-2 expression which represents an osteogenic
  stimulus.
• Similarly, statins increased the number of mouse
  osteoblasts and the amount of new bone formed,
  similar to that seen with recombinant BMP-2
  itself.

Mundy G et al, Science 1999
67

• However, current attempts including the ongoing
  ASTRONOMER trial will help to further resolve
  this issue and clarify the role of statins in the
  prevention of cardiovascular disease progression.

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Biphosphonates

• In a retrospective review of patients followed
  for mild or moderate AS, these investigators
  found that 18 patients receiving treatment for
  osteoporosis had significantly less decrement in
  aortic valve area on follow-up echocardiography
  than 37 not receiving such treatment.
• The most attractive explanation is an action of
  drug therapy for osteoporosis, most often
  bisphosphonates, to retard aortic valve
  calcification.
• The mechanism for this action is not clear,
  although numerous possibilities can be postulated
  on the basis of the multiple complex processes
  controlling tissue calcification.

Skolnick et al Am J Cardiol 2009
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• In an animal model of induced arterial and
  valvular calcification, bisphosphonate doses
  producing inhibition of bone resorption were also
  effective in preventing cardiovascular calcium
  deposits, with no accompanying changes in serum
  calcium or phosphorus.

Price PA Arterioscler Thromb Vasc Biol 2008
70
 Proportion (percentage) of patients on OT
stratified by slow progression (lt0.1 cm2/year),
moderate progression (0.1 to 0.2 cm2/year), and
rapid progression (gt0.2 cm2/year) of AS.
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Fluvastatin, significantly attenuated osteoclast
differentiation and activation through a blockade
of the classical mevalonate pathway and an
antioxidant action, leading to prevention of
osteoporosis.
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Effect of atorvastatin on bone mineral density in
patients with acute coronary syndrome. Pérez-Castr
illón JL, Abad L, Vega G, Sanz-Cantalapiedra A,
García-Porrero M, Pinacho F, Dueñas A.
CONCLUSION Atorvastatin has a beneficial effect
on bone metabolism in patients with acute
ischemic heart disease (mainly males) by
incrementing bone mineral density in which
vitamin D levels are required to be higher than
30 nmol/l for the drug to be effective.
Eur Rev Med Pharmacol Sci.
73
ACE Inhibition
Rate of hemodynamic progression of AS in
normotensive patients, hypertensive patients
receiving ACEI therapy, and hypertensive patients
not receiving ACEI therapy.
Rosenhek R, Circulation 2004
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Schematic view of risk factors and mediators
which play a role in the pathogenesis of aortic
valve calcification.
Yetkin E, Int J Cardiol 2009
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Conclusions

• Although there are similarities with the process
  of atherogenesis as well as with some risk
  factors , not all the patients with coronary
  artery disease or atherocsclerosis develop
  calcific aortic stenosis.
• So there must be some differentiating process.
• Most of the patients who have the known risk
  factor such as diabetes mellitus,
  hypercholesterolemia or hypertension, have
  already been treated or have been taking drugs
  such as HMCoA reductase inhibitors or ACE
  inhibitors.

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• Dissapointing results from the randomized trials
  of statin treatment necessitate novel treatment
  modalities.
• Suppressing the inflammatory process and
  preventing osteoblastic differentiation in valve
  tissue seem to be potential novel strategies for
  preventing the progression of calcified aortic
  stenosis.

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Thanks for your attention