Heart Valve Lesions Due to the Formation of a Beta-Hemolytic Streptococ Role of Adhesion Molecules

doi:10.4236/ss.2013.48073

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Surgical Science, 2013, 4, 371-376

http://dx.doi.org/10.4236/ss.2013.48073 Published Online August 2013 (http://www.scirp.org/journal/ss)

Heart Valve Lesions Due to the Formation of a

Beta-Hemolytic Streptococ Role of Adhesion Molecules

Semsi Altaner1, Turhan Kurum2, Muzaffer Demir3, Burhan Turgut4, Turan Ege5, Enver Duran5

1Pathology Department, School of Medicine, Baskent University, Istanbul, Turkey

2TEM Hospital, Istanbul, Turkey

3 Hematology Department, School of Medicine, Trakya University, Edirne, Turkey

4 School of Medicine, Namik Kemal University, Tekirdag, Turkey

5School of Medicine, Trakya University, Edirne, Turkey

Email: [email protected]

Received June 8, 2013; revised July 9, 2013; accepted July 18, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Objectives: A rise in the levels of adhesion molecules such as VCAM-1, ICAM-1and E-selectin in valve disease pa-

tients has been reported lately. In our study, by detecting the presence of adhesion molecule expression in the valve en-

dothelium we will try to show the level of adhesion molecules in peripheral blood leucocytes. Materials and Methods:

Valve samples were obtained from patients having undergone aortic and mitral valve replacement due to symptomatic

aortic stenosis/aortic insufficiency and/or mitral stenosis/mitral insufficiency. The clinical preoperative diagnosis was

made using two-dimensional echocardiography and Doppler echocardiography. Rheumatic valves were in group B (n =

20). Group A (n = 8) constituted the control group. Immunohistochemical staining was performed using CD4, CD8,

CD54/ICAM-1, and CD106/VCAM-1. Flow cytometric analysis was performed. The Kolmogorov-Smirnov test and

Fisher’s exact test were used for the comparison of categorical variables. Results: Group A (non-rheumatic) patients

were found to be older than group B (rheumatic) patients (59.8 ± 11.4 years vs. 45.3 ± 11.8 years, p = 0.008). In group

B VCAM-1 level was higher than that of group A (296.6 ± 21.2 vs. 258.5 ± 42.1, p = 0.004). CD11b monocyte in group

B was higher than in group A (98.8 ± 0.5 vs. 92.9 ± 9.7, p = 0.003). CD11b granulocyte was higher in group B than in

group A (99.96 ± 0.05 vs. 93.79 ± 13.26, p = 0.33). Significant differences were not determined in the other parameters.

Conclusion: The fact that increases in serum VCAM-1 and CD-11b only occurred in patients with rheumatic valvular

disease in our study suggests that inflammation in patients with the same hemodynamic disorder is higher in rheumatic

valvular disease than in the ones with non-rheumatic valvular disease.

Keywords: Rheumatic Fever; Chronic Valve Disease; Cell Adhesion Molecules

1. Introduction

Rheumatic fever (RF) is an inflammatory disease where

the immune mechanism targets the heart following Group

A beta-hemolytic streptococcal pharyngitis [1,2]. Im-

mune response developing against streptococcal anti-

gens in the development of RF has been accepted as a

cause of initiation of the disease. Both genetic and envi-

ronmental factors are effective in the development of RF.

RF mostly reveals itself by leading to carditis. Pathologi-

cal signs of rheumatic carditis, which include Anitschkow

myocytes and Aschoff nodules, have an effect on heart

tissue [3,4]. Valve involvement is diagnosed by observ-

ing the development of verru and nodules on the mitral

valve, accompanied by edema and cellular inflamma-

tion.

The approach accepted during the past half-century

has appeared to be that Group A streptococcal antigens

may break immune tolerance in vivo as a consequence of

antigenic similarity and/or an abnormal response to this

bacterial antigen [5]. Although factors that precede the

progression of fibrosis in some patients with rheumatic

fever have not been entirely revealed, progressive fibro-

sis of valves and chronic valve disease develop in such

patients. The pathogenetic mechanisms causing RF and

non-rheumatic heart disease could still not be entirely

determined [5].

Rheumatic heart disease is the most frequent cause of

valvular diseases in developing countries [5]. Rheumatic

fever and non-rheumatic heart diseases are still an im-

portant worldwide public health problem in terms of car-

S. ALTANER ET AL.

372

diovascular mortality and morbidity in the 21st century

[6]. Chronic rheumatic valvular disease is considered to

be a late sequela that occurs in 30% of patients with

rheumatic fever. Mitral valvulitis is the most common

lesion of acute rheumatic carditis. Valvulitis generally

occurs in the valves and chordae tendineae due to fibrous

tissue accumulation as a consequence of recovery and

disease episodes. Rheumatic fever is diagnosed in 99%

of patients who undergo mitral valve replacement due to

mitral valve stenosis [6].

There is a period of at least 2 years and generally long

intervals (10 to 20 years) between the occurrence of

rheumatic carditis attack and symptomatic valve disease

[7,8]. Primary lesions of the valve in RF patients are

translucent nodules observed along the closure lines of

the valve. The valve weakens and has a fibrotic and cal-

cified appearance due to the development of valvulitis.

Mitral valvulitis occurring due to an attack of RF causes

abnormal flow across the valve. This changing flow pat-

tern develops an increasing tension on the disordered

structure of the valve. In addition, inflammation in the

valve leads to the development of fibrin deposits on the

surface of the valve which causes an increase in the ab-

normal flow pattern across the valve. Tension and pres-

sure exerted over the course of years results in weakness

and fibrosis of the valve. Anatomical differences in pa-

tients of severe mitral stenosis (MS) or aortic stenosis

(AS) are developed by the stable and progressive trauma

caused by the turbulent flow [9]. It is still discussed

whether the development of anatomical differences in the

valve are of a progressing rheumatic origin or progress-

sive fibrosis, weakening and calcification developing in

the valve which is deformed by a turbulent flow. The

endothelial cells in the walls of large vessels, such as the

aorta, and cardiac valves are exposed to high shear stress.

Cardiac valve endothelial cells have a special morpho-

logical structure. These valves are in the form of surface

microvilli [10].

Cell adhesion molecules are special structures found

on the surface of circulating leucocytes and endothelial

cells, which regulate the migration of leucocytes to the

functionally different body tissues. These cell adhesion

molecules provide the orientation of effectors cells to the

sites of inflammation and tissue destruction, lymphocyte

circulation among lymphoid tissues and the progenitor

cell development and maturity within the hematopoietic

microenvironment [11,12]. Cell adhesion molecules in

human serum in healthy patients can be detected at very

low levels. However, there is also a group of diseases

with inflammatory or vascular etiology for which low

levels of cell adhesion molecules can be detected.

Intercellular adhesion molecule-l (ICAM-1), vascular

adhesion molecule-1 (VCAM-1) and E-selectin are

structures functioning as receptors for the inflammatory

cells which are detected circulating on the vascular en-

dothelium. ICAM is a membrane glycoprotein which

allows glycolysis at different values [13,14]. Endothelial

cells are situated on the fibroblasts, epithelial cells and

active lymphocytes. VCAM-1 are structures activated by

Interleukin-1B (IL), TNF-alpha and IL-4 situated primar-

ily on bone marrow fibroblasts, macrophages, synovial

lining cells, germinal center dendritic cells and endothe-

lial cells [15]. Selectins are structures which have a very

important role in the first contact and rolling of leuco-

cytes on the active endothelium. E-selectin is a molecule

found on endothelial cells stimulated only by cytokines

[13,14]. The increased serum levels of this molecule are

accepted as an indicator of endothelial cell activation.

Unlike other adhesion molecules which have a wider

tissue distribution, E-selectin is found only on the acti-

vated endothelium. A rise in E-selectin level will show

endothelial activation as a component of a special pa-

thology or damage. In vivo expression generally appears

along with active granulomatous inflammation and de-

layed hypersensitivity.

CD11b (Mac-1 α chain) is one of the β 2-integrins ex-

pressed on leucocytes. It mediates leukocyte adhesion

and transmigration to the endothelial cells. In addition, it

has a role in neutrophil adhesion (homotopical adhesion)

and chemotaxis.

A rise in adhesion molecule levels, such as VCAM-1,

ICAM-1and E-selectin in valve disease patients has been

reported recently [16-18]. In our study by detecting the

presence of CD11b lymphocytes in the valve tissue, ad-

hesion molecule expression in the valve endothelium and

the level of adhesion molecules in peripheral blood leu-

cocytes, we will try to show whether there is an active

inflammatory process during the chronic period of rheu-

matic fever. On the condition of the presence of the

process we will try to differentiate whether a progressive

inflammation or hemodynamic loading caused by under-

lying preload and after load increase leads to an increase

in adhesion molecules by applying the findings at tissue

level.

2. Materials and Methods

2.1. Tissues

Valve samples were obtained from patients having un-

dergone aortic and mitral valve replacement due to sy-

mptomatic aortic stenosis/aortic insufficiency and/or mi-

tral stenosis/mitral insufficiency in Trakya University

Hospital for histopathological examination. The clinical

preoperative diagnosis was made using two-dimensional

echocardiography and Doppler echocardiography. Calci-

fied and/or fibrotic rheumatic valves were collected dur-

ing the operations. (Group B; n = 20, 11 male, 9 female).

Mitral valve tissue evaluated as ischemic during valve

S. ALTANER ET AL. 373

repair surgery and aortic valve tissue of non-rheumatic

etiology (Group A; n = 8, 5 male, 3 female) constituted

the control group. Patients with malignity, inflammatory

disease, renal or liver disease, diabetes mellitus, hyper-

tension, hyperlipidemia, deep venous thrombosis, pul-

monary emboli or a past history of surgery in both

groups were excluded from the study. Whether or not

calcification was present, commissural fusion and fibrous

thickening within the site of valve closures and revascu-

larization were evaluated as the macroscopic evidence of

rheumatic origin.

2.2. Immunohistochemical Study

All tissue samples were fixed with 10% neutral formalin

and embedded in paraffin. 3-µm thick sections were ob-

tained from paraffin blocks. First, the sections were de-

hydrated in series of alcohol then they were placed in a

10 mM citrate tampon and were exposed to antigen re-

trieval for 40 minutes. After heating, the sections were

left to cool at room temperature and they were washed

with tampon saline. They were treated with 3% hydrogen

peroxide in methanol for 5 minutes. Immunohistochemi-

cal marking was performed using a streptavidin-biotin

peroxidase system (DAKO LSAB kit, United Kingdom).

CD4 Ab-8 (clone 4B12, Neomarkers, United Kingdom),

CD8 Ab-7 (clone 4B11, Neomarkers, United Kingdom),

CD54/ICAM-1 Ab-4 (Clone 54CO4, Neomarkers, United

Kingdom), and CD106/VCAM-1 Ab-3 (Clone 1.4 C3,

NeoMarkers, United Kingdom) were incubated for an

hour at room temperature. Secondary antibodies were

performed for 15 minutes after rinsing with a saline

phosphate tampon. The sections were incubated with

streptavidin-biotin complex for 10 minutes and treated

for 5 minutes with a hydrogen peroxide phosphate tam-

pon. They were colored with 3.3 diaminobenzidine tet-

rahydrochloride (DAB). Mayer’s hematoxylin was per-

formed as counterstaining. Afterwards, the sections were

dehydrated with a series of ethanol performance. Cyto-

plasmatic and membranous staining was accepted as

positive for ICAM-1 and VCAM-1.

2.3. Cytokins

Blood samples were obtained in the fasting state between

9 and 10 am in the control and study groups in order to

be able to exclude the possible influence of circadian

fluctuation. Blood samples were collected in 5-ml tubes

containing 3.8% Na-citrate. Blood samples were ob-

tained under minimal tourniquet pressure from the ante-

cubital vein using a sterile 22-gauge needle syringe in a

single attempt, from the patients in the supine position

for at least 20 minutes and the samples were centrifuged

at 3000 turnover for 10 minutes at +4˚C. Plasma was

decomposed and frozen at −80˚C. Blood samples were

analyzed according to standard laboratory methods in-

cluding whole blood count, biochemical and electrolyte

measurements, and blood samples with excessive hemo-

lysis were excluded from the study. The soluble VCAM-

1 and ICAM-1 concentrations of human plasma samples

were measured by using commercially available, solid-

phase, enzyme-linked kits (Diaclone; Besancon, France).

The blood samples were processed according to the in-

structions of the manufacturer. The duration between the

collection of blood samples and antibody labeling was

standardized as an hour. Anti-VCAM-1 and anti-ICAM-1

antibodies were sequentially added after the testing sam-

ples were treated with monoclonal antibodies found in

the standard of known soluble concentrations of VCAM-

1 and ICAM-1. Unbound materials were removed and an

enzyme (streptavidin-peroxidase) was added after an

hour of incubation at room temperature. Subsequent to

incubation and washing to remove the entire unbound

enzyme, a substrate solution (chromogen [tetramethyl-

benzidine]) was added to induce a color reaction. The

color reaction was measured using a microplate reader

(Dynex Technologies; Chantilly, UK) and reading the

absorbance at 450 nm with a correction wavelength of

630 nm. A standard curve was obtained using the mean

absorbance values of VCAM-1 and ICAM-1, and the

VCAM-1 and ICAM-1 concentrations in unknown

plasma samples were calculated by linear regression. All

standards and samples were tested in duplicate. Accord-

ing to the manufacturer of the kits, the minimum quanti-

fiable doses of VCAM-1 and ICAM-1 are <0.6 ng/ml

and <0.1 ng/ml respectively.

2.4. Cell Preparation, Marking and Flow

Cytometric Analysis

Fluorescein isothiocyanate (FITC), phycoerythrin (PE)

and phycoerythrin-cyanin 5.1 (PC5) dyes with conjuge

monoclonal antibodies isotophic controls were used in

the study carried out with three colors through direct

immunofluorescence method.

Preparing the samples: The blood sample with 100 μl

EDTA was treated in a suitable way and incubated with a

monoclonal antibody cocktail at room temperature and in

darkness for 20 minutes. In addition to this, lysis of

erythrocytes and fixation of leukocytes were carried out

in a multi-Q prep workstation, Beckman Coulter, which

was an automated device for preparing samples. Analysis

was conducted using a coulter Epics XL current cytome-

try device. Expression rates of antigens on lymphocytes,

determined according to light characteristics formed on

the histogram with system II software, were measured.

2.5. Statistics

Continuous variables were described in terms of the

S. ALTANER ET AL.

374

mean ± SD and categorical variables were described as

percentages. The suitability of the continuous variables

with normal distribution was examined using the Kol-

mogorov-Smirnov test. In comparing variables between

the groups, a T-test was used for independent groups

with normal distribution but the Mann-Whitney U test

was used for independent groups without normal distri-

bution. Fisher’s exact test was used for the comparison of

categorical variables. P < 0.05 was considered statistic-

cally significant.

3. Results

The clinical characteristics for each patient are summa-

rized in Table 1. Group A patients were found to be

older than group B patients (59.8 ± 11.4 years vs. 45.3 ±

11.8 years, p = 0.008). In group B the ICAM-1 level was

higher than that of group A (296.6 ± 21.2 vs. 258.5 ±

42.1, p = 0.004). Group B had higher monocyte CD11b

levels in group A (98.8 ± 0.5 vs. 92.9 ± 9.7, p = 0.003).

CD11b granulocyte level was higher in group B group

than in group A (99.96 ± 0.05 vs. 93.79 ± 13.26, p =

0.33). Significant differences were not determined in

other parameters. No significant correlation between the

parameters of RVH and non-RVH could be found with

immunohistochemistry. No difference was found be-

tween gender and the diagnosis of RVH and non-RVH.

The light microscopic examination with hematoxylin

eosin revealed that the valves were observed to consist of

fibro-hyalinized tissues in wide sites. Some of the heart

valves exhibited histiocyte and lymphocyte infiltration

and in some of them young connective tissue prolifera-

tion existed. In all heart valves there were small-sized

vascular structures which were not so intense. There

were no differences in both groups at the light micro-

scopic level.

In immunohistochemical studies, CD4 or CD8 cyto-

plasmic positivity was detected in some of the lympho-

cytes, making clusters in patches located in valvular

stroma. In some lymphocytes, fibroblasts and endothelial

cells, rare CD54 (ICAM-1) cytoplasmic positivity was

detected. CD106 (VCAM-1) positivity was observed in

vascular endothelial cells and some fibroblasts.

4. Discussion

Rheumatic fever is a multi-systemic inflammatory dis-

ease occurring following group A beta-hemolytic strep

Table 1. CD4, CD8, ICAM-1 and VCAM-1 distribution of

non-rhe umatic and rh e umatic groups.

CD4 CD8 ICAM-1VCAM-1

(Group A (non-rheumatic) (n = 8) 1 1 1 2

Group B (rheumatic) (n = 20) 7 5 5 10

toccal pharangitis and it commonly affects the heart. It is

also triggered by immunological mechanisms. Recurrent

streptococcal pharyngeal infections after a previous at-

tack increases the reactivation risk of the disease. The

number of lymphocytes causes an increase both in car-

diac valves and in peripheral blood and also the cytokine

levels in blood increase during acute rheumatic carditis.

In this course, adhesion molecules play a considerable

role. Intracellular adhesion molecules (ICAM-1), vascu-

lar cell adhesion molecules (VCAM-1) and E-selectin

exist in the vascular endothelium and act as ligands for

surface receptors of circulating inflammatory cells [11,

13-15].

It is known that adhesion molecules differentiate their

antigenic expression according to their activation status

and location [19,20]. Detailed pathological investigations

researching the circulating forms of VCAM-1, ICAM-1

and E-selectin in the plasma have revealed the increased

expression of cellular adhesion molecules in endothelial

cells and other tissue types [21-23].

Yetkin et al. compared 34 patients who underwent

balloon mitral valvuloplasty with 34 healthy people par-

ticipating as the control group [18]. In their study the

serum levels of E-selectin, ICAM-1 and VCAM-1 were

found to be elevated in patients with mitral stenosis

compared with the control group. According to this result,

they proposed that E-selectin, ICAM-1 and VCAM-1,

which were the elevated serum adhesion molecules, are

indicators of ongoing chronic inflammation in patients

with significant rheumatic mitral stenosis. The research-

ers subsequently examined the values in the patient

group before and after the procedure and found that after

PMBV procedure, VCAM values were elevated, E-se-

lectin values had decreased and ICAM remained un-

changed. They proposed with respect to this result that

ICAM-1, VCAM-1 and E-selectin exhibit discrete

changes as a response after percutaneous mitral balloon

valvuloplasty [24]. They indicated that further investiga-

tions should be required to clarify the mechanism of the

relationship between adhesion molecules and PMBV in

addition to rheumatic mitral stenosis. Chen et al. pro-

posed that serum VCAM-1 level decreased significantly

after PMBV and elevated plasma VCAM-1 level was

related to hemodynamic abnormality, not rheumatic ac-

tivity [25].

Shahi et al. determined that soluble adhesion mole-

cules such as ICAM-1, VCAM-1 and E-selectin are ele-

vated in non-rheumatic aortic valvular stenoses. The au-

thors proposed that the pathogenesis of non-rheumatic

aortic stenosis which had been believed to be degenera-

tive actually had an inflammatory and immunologic eti-

ology. No study investigating the tissue and serum levels

of cell adhesion molecules of patients with rheumatic

aortic valvular disease was detected in literature.

S. ALTANER ET AL. 375

In the above mentioned studies because no immuno-

chemical studies were performed on the valvular tissues

of patients with mitral or aortic valvular diseases, and

because the relationship between serum levels was not

investigated, it could not established whether these find-

ings resulted from hemodynamic disorders or from de-

genaration. We determined that VCAM-1 in serum levels

increased in patients with rheumatic valvular disease

compared to the ones with non-rheumatic valvular dis-

ease. No significant difference was found in other adhe-

sion molecules from the serum examination. Hence, we

assume that hemodynamic disorders may have an in-

creasing effect on cell adhesion molecules in addition to

inflammation.

Roberts et al. tried to clarify the immunity-mediated

mechanism in rheumatic heart disease caused by group A

streptococcal infection by analyzing anti-CD4, anti-CD8

and VCAM-1 in valvular tissues acquired from 6 patients

with rheumatic valvular disease requiring valvular re-

placement (2). Out of the 6 patients, only 2 had an endo-

thelium suitable for examination. They detected that CD4

and CD8 lymphocytes penetrated through the subendo-

thelial layer on the valve surface and attached to the

valve endothelium. They proposed that T cell extravasa-

tion through the surface endothelium into the valve

seemed to be an important process in rheumatic heart

disease development. The researchers anticipate that

lymphocytic infiltration through valve surface endothe-

lium is a potential triggering step in disease pathogenesis.

However, there was no information about the serum val-

ues of the patients in this study. Mitral valves taken from

3 in order to control autopsy subjects were studied. As

for our study in the same patient group with mitral steno-

sis and/or mitral failure and/or aort stenosis and/or aort

failure, examination was performed for adhesion mole-

cules both in valvular tissue and soluble adhesion mole-

cules in blood. However, in our study, ICAM-1, VCAM-

1 and E-selectin were analyzed using IHK method both

in patients with ischemic and degenerative valvular dis-

ease and in the ones with rheumatic valvular disease, and

no significant difference was found.

Ghaisas et al. studied ICAM-1, VCAM-1 and E-se-

lectin expression via immunohistochemical method in

patients with non-rheumatic aortic disease requiring aor-

tic valvular replacement. They also analyzed the serum

values before and after surgery. They reported that adhe-

sion molecules located in the tissues increased. E-se-

lectin increased both in serum and tissue. As a cones-

quence, they proposed that the expression of adhesion

molecules in the involved valves supported the inflame-

matory component theory in degenerative aortic valvular

disease and this was the main source for E-selectin lo-

cated in the serum [17].

Muller et al. investigated the expression of cell adhe-

sion molecules using immunohistochemical method in

degenerative cardiac valves and the endothelial cells of

heart valves with endocarditis [26]. As expected, ICAM-

1, CD34 and CD31 were found both in degenerated and

inflamed valves. Muller et al. detected that significant

expressions of E-selectin and VCAM-1 were not only in

inflamed cardiac valves but also in the majority of de-

generated valves not exhibiting the morphological signs

of inflammation. They thought that they could present

this as an attractive finding to explain why fibrotic car-

diac valves were prone to recurrent endocarditis. Also,

they proposed that further investigation was required to

discover why E-selectin and VCAM-1 which are endo-

thelial activation markers were also expressed in degen-

erated cardiac valves [26].

Wallby et al. investigated T lymphocyte infiltration by

comparing the patients with degenerative calcification of

tricuspid aortic valves to patients with congenital bicus-

pid aortic valve secondary calcification [27]. Conse-

quently without considering valvular anomaly, by deter-

mining T lymphocyte infiltration in acquired aortic

stenosis equally, they proposed that T lymphocyte infil-

tration should be regarded in inflammation pathogenesis.

Finally, none of these studies examined or compared

rheumatic and non-rheumatic valvular disease in terms of

serum and tissue values. That only serum VCAM-1 and

tissue CD11b increases occurred in our study in patients

with rheumatic valvular disease suggests that inflamma-

tion in patients with the same hemodynamic disorder is

higher in rheumatic valvular disease than in the ones with

non-rheumatic valvular disease.

Restrictions: The values for rheumatic and ischemic

valvular diseases were not compared with the normal

control group. Average values were above the cut-off

level. In addition, control values were not examined after

valvular changes. For this reason, whether hemodynamic

improvement occurred or not cannot be shown with the

serum values.

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