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(Circulation. 1995;91:275-283.)
© 1995 American Heart Association, Inc.

Articles

Increase and Redistribution of Cardiac Mast Cells in Auricular Thrombosis

Possible Role of kit Ligand

Hans C. Bankl, MD; Thaddäus Radaszkiewicz, MD; Günter W. Klappacher, MD; Dietmar Glogar, MD; Wolfgang R. Sperr, MD; Karl Großschmidt, MD; Hans Bankl, MD; Klaus Lechner, MD; Peter Valent, MD

From the Department of Internal Medicine I, Division of Hematology and Hemostaseology, Department of Internal Medicine II, Division of Cardiology, Histological and Embryological Institute, Clinical Institute for Medical and Chemical Laboratory Diagnostics and Department of Clinical Pathology, University of Vienna, Austria.

Correspondence to Hans C. Bankl, MD, Department of Internal Medicine I, Division of Hematology, University of Vienna; Währinger Gürtel 18-20, A-1090 Vienna, Austria.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background The atrial appendage is a predilection site for thrombus formation. Mast cells (MC) are a rich source of mediators that may be involved in the regulation of thrombus formation. We examined number, distribution, and phenotype of MC in thrombosed versus unaffected auricles to elucidate their possible role in auricular thrombosis (AUTHR).

Methods and Results Sections of atrial appendages (AUTHR, n=14; controls (CO), n=13) were analyzed for MC by Giemsa, toluidine blue, and berberine sulfate stains and by immunohistochemistry. Cardiac MC expressed CD antigens corresponding to the classic MC phenotype as well as tryptase, chymase, and heparin. Thrombosis was associated with a twofold increase in the number of MC in the total appendage (CO, 3.1±1.0 versus AUTHR, 6.4±1.1 MC/mm², P<.01). Moreover, in AUTHR, a redistribution of MC to the upper endocardium was observed (AUTHR, 5.3±1.4 versus CO, 0.07±0.15 MC/mm², P<.01). Mast cell growth factor (MGF) was expressed in the endothelium and subendothelial space of thrombosed appendages but not in the normal endocardium. Overexpression of MGF was accompanied by a weak or absent expression of the MGF receptor c-kit on redistributed MC in AUTHR. Patients with unilateral atrial appendage thrombosis did not exhibit a MC increase or redistribution in the unaffected contralateral appendage. No augmentation of other inflammatory cells was observed. Stimulation of isolated cardiac MC with MGF resulted in mediator release.

Conclusions This study provides evidence that AUTHR is associated with MC increase and redistribution and MGF overexpression. The role of redistributed MC and their mediators in the pathophysiology of atrial thrombosis requires further investigation.

Key Words: thrombosis • cells • receptors

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
The predilection of the atrial appendage for thrombus formation has long been known.¹ However, the pathogenesis of auricular thrombosis has not been entirely specified yet. Virchow stated the triad of stasis of blood flow, altered characteristics of the blood, and vessel wall injury. The increased incidence of thrombus formation in atrial fibrillation implies a role for hemodynamic factors. Pollick and Taylor² demonstrated that atrial thrombus formation is associated with decreased atrial appendage contraction and atrial appendage dilation. The involvement of humoral factors in thrombogenesis is supported by the fact that anticoagulant therapy in atrial fibrillation lowers the risk of stroke.^{3 4} Endothelial and subendothelial cells also may contribute to local thrombus formation due to their active role in the regulation of blood coagulation.⁵ So far, the exact molecular mechanisms and cell types involved could not be defined.

Mast cells (MC) are multifunctional immune cells in loose connective tissue usually located in vicinity to small blood vessels.^{6 7 8} Human MC produce a number of vasoactive or thromboactive mediators including histamine, heparin, proteolytic enzymes, or cytokines.^{9 10 11 12} Some of these compounds, such as heparin, tryptase, or chymase, are almost exclusively expressed in MC. Growth and function, ie, mediator production and secretion, of MC are regulated by mast cell growth factor (MGF),^{13 14 15 16} also called stem cell factor (SCF) or kit ligand.^{17 18} This cytokine represents the ligand of the c-kit tyrosine kinase receptor recently clustered as CD117.¹⁹

The purpose of this study was to examine the number, distribution, and phenotype of MC in thrombotic atrial appendages and to evaluate differences to unaffected auricles in order to elucidate a possible role of MC and their mediators in auricular thrombosis (AUTHR).

	Methods

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Patients and Tissue Processing
Myocardial tissue was obtained from 24 patients at autopsy and from three explanted hearts after cardiac transplantation (Table 1). Tissue samples from two different locations were taken from each auricle. Sites of adherent organized thrombi were not evaluated since organization destroyed preformed structures. After resection, tissue was cut and divided, and one part was fixed in neutral buffered formalin (8% formalin in 0.08 mol/L sodium phosphate, pH 7.4) and the other part in Carnoy's fluid (60% ethanol, 30% chloroform, 10% glacial acetic acid). Tissue was then embedded in paraffin and cut into sections of 2 µm. Additionally, tissue was snap-frozen in precooled isopentane and prepared for cryostat sections.

View this table: [in this window] [in a new window]   Table 1. Patient Characteristics

Monoclonal Antibodies
A series of monoclonal antibodies (mAbs) was used for immunohistochemical analysis: antichymase and antitryptase both purchased from Chemicon, mAb 1.D9.3D6 (anti–c-kit) from Boehringer Mannheim, mAb 1D5.E11/IgG1 (anti–c-kit) from Cedarlane, anti–membrane-bound SCF from Genzyme, mAbs anti–₁-antitrypsin, anti-₁-antichymotrypsin, antilysozyme, antimyeloperoxidase, anti-CD3, anti-LCA, Y2/51 (anti-CD61), KP-1, PG-M1 (both anti-CD68), and L26 from Dako, mAb BA2 (anti-CD9) from Jansen, anti–PECAM-1 from British Biotechnology, anti–ELAM-1 from Cambridge Research Biochemicals, mAbs 84H10 (anti–ICAM-1), 1G11 (anti–VCAM-1), and E124.2.8 (anti-IgE) from Immunotech, mAb 4B4 (anti-CD29) and mAb My9 (anti-CD33) from Coulter, and mAbs Leu M-1, Leu 15 (anti-CD11b), Leu 22 (anti-CD43), and Leu 44 (anti-CD44) from Becton Dickinson. The mAbs 1A2.C5, 1D5.E11/IgM, 95C3 (all anti–c-kit), and 4B10 (anti–membrane-bound SCF) were obtained from the Fifth International Workshop and Conference on Human Leukocyte Differentiation Antigens (Boston, 1993).¹⁹ The anti–c-kit mAb YB5.B8 was kindly provided by L.K. Ashman (University of Adelaide, Australia).²⁰ Specifications of mAbs used in this study are summarized in Table 2.

View this table: [in this window] [in a new window]   Table 2. Specification of mAbs Used in This Study

In Situ Staining Techniques
Immunohistochemistry
Immunohistochemistry was performed according to Hsu et al.²¹ In brief, cryostat sections were thawed, dried, and fixed in acetone or methanol for 10 minutes. Paraffin-embedded sections were dewaxed, and the endogenous peroxidase was blocked with 5% H₂O₂ in methanol for 15 minutes. After each step, the tissue was rinsed twice in Tris-buffered saline (TBS) (pH 7.6). Slides were treated with TBS, 0.1% protease type XIV (Sigma) for 10 minutes. Nonspecific binding was blocked with TBS, 1% horse serum (Vector). Primary mAbs were diluted in TBS, 1% horse serum, and applied for 60 minutes. Next, sections were incubated with a biotinylated horse anti-mouse IgG (Vector) for 30 minutes and either streptavidin-biotin-peroxidase or streptavidin-biotin-alkaline phosphatase complexes (Dako). Diaminobenzidin (DAB) (Vector) was used as chromogen, giving a brown or black reaction product with horseradish-peroxidase. Neofuchsin (Dako) was used for alkaline-phosphatase detection, providing a red stain. Sections were counterstained in Gill's hematoxylin. Control slides were equally treated either with the primary antibody omitted or using isotype matched mAbs or normal rabbit serum.

Double Staining Techniques
Sequential and simultaneous double-labeling immunohistochemistry was performed essentially as described by Irani et al.²² For sequential staining, sections were first incubated with primary mAb, then with secondary biotinylated horse anti-mouse IgG and next with FITC-labeled streptavidin (Vector). Thereafter, an alkaline phosphatase-conjugated antibody for the detection of the second antigen was applied. The reaction was visualized by fast blue salt (Vector), resulting in a blue color. For simultaneous double labeling, tissue sections were incubated with a mixture of a biotinylated and an alkaline phosphatase-conjugated antibody. Cells reacting with alkaline phosphatase-conjugated antibody stained blue by addition of fast blue salt and reactivity of the biotinylated antibody was visualized by either FITC-streptavidin or peroxidase-conjugated streptavidin and DAB. Specificity of each step was proved by the use of isotype control mAbs.

Toluidine Blue Staining
Deparaffinated sections were stained with 1% toluidine blue (Merck) in methanol (pH 7.4) for 5 minutes, washed for 2 minutes, differentiated in 0.1% acetic acid in methanol, and mounted after air-drying.

Berberine Sulfate Staining
Berberine sulfate staining was performed as described previously.²³ In brief, deparaffinated sections were fixed in ethanol/acetic acid (3:1) for 15 minutes and washed in ethanol and in water before staining with 0.02% berberine sulfate (Serva) in water with 1% citric acid (pH 4.0) for 20 minutes. Subsequently, the slides were washed in water with 1% citric acid (pH 4.0), dried, and mounted in Entellan (Merck). In addition, adjacent sections were pretreated with 10 U/mL heparinase (Sigma) in TBS (pH 7.0) or with TBS alone for 90 minutes at 37°C.²⁴

Determination of Mast Cell Numbers in Tissue Sections
To determine the number of MC in endomyocardial regions, paraffin-embedded material stained with either Giemsa or immunohistochemistry (tryptase, chymase, c-kit) was analyzed. Stained sections were scanned into a personal computer (IBM). The total endocardial and myocardial areas in a given section were marked, and the size of areas was calculated by computer (Vistatips, AT&T). The total number of MC in a given section as well as in each region were counted under light microscopy at x400 magnification by two independent observers. The total number of MC was calculated as cells/mm².

Isolation of Cardiac Mast Cell and Histamine Release Experiments
To assess the content of mediator substances and their releasability from cardiac MC after stimulation with MGF histamine release experiments were performed. Human cardiac MC were isolated from three atrial appendages after cardiac transplantation by enzymatic digestion, as described previously.²⁵ In brief, tissue was incubated with collagenase (30 U/mL) (Worthington) for 2 hours at 37°C. After filtration and washing in 0.9% NaCl, the cell suspension was incubated with DNAse (0.5 mg/mL), hyaluronidase (0.5 mg/mL), and pronase-E (2 mg/mL) (all from Sigma) for 15 minutes at 37°C to remove myocyte cell ghosts. The presence of MC was determined by toluidine blue and Giemsa staining. Isolated MC were cultured in RPMI-1640 medium with 10% FCS for at least 24 hours before being analyzed. Histamine release was performed on cardiac MC according to published techniques.²⁵ MC were incubated with various concentrations of recombinant human MGF (Genzyme) for 60 minutes at 37°C. After centrifugation at 4°C, cell-free supernatants were recovered and analyzed. Total histamine was determined in cell lysates after freeze-thawing. Histamine was measured by a commercial radioimmunoassay (Immunotech). Histamine release was calculated as percent of total histamine.

Statistical Evaluation of Data
Differences in MC number and phenotype were analyzed by standard tests including the Student's t test and linear regression and correlation. A P value of <.05 was considered statistically significant. Values represent mean±SEM.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Localization, Number, and Distribution of Cardiac Mast Cells in Auricular Thrombosis
MC were detected in the epicardium, in the myocardium, and in the endocardium of atrial appendages by Giemsa, toluidine blue, and immunohistochemistry with mAbs to MC antigens. We found a twofold increase of MC numbers in thrombosed auricles when compared with unaffected control appendages (CO) (CO, 3.1±1.0 versus AUTHR, 6.4±1.1 MC/mm², P<.01). MC concentration increased significantly in each layer: in the myocardium (CO, 3.1±1.0 [range, 1.2 to 4.7] versus AUTHR, 5.9±0.9 MC/mm² [range, 4.1 to 7.2], P<.01) as well as in the total endocardium (CO, 3.2±1.5 [range, 1.0 to 5.8] versus AUTHR, 7.3±1.7 MC/mm² [range, 4.3 to 9.9], P<.01) (Fig 1). A thorough examination of the distribution of MC revealed that MC redistribute to the upper endocardium in thrombosed atrial appendages, whereas no substantial amounts of MC could be detected in the subendocardial region of unaffected appendages. No redistribution of MC was observed in the epicardium and myocardium of AUTHR versus CO. More than 90% of the upper endocardial MC in auricular thrombosis were located in close vicinity (<50 µm) to the endothelium and the thrombus (Fig 2). Evaluation of MC concentration in the upper endocardium revealed CO, 0.07±0.15 versus AUTHR, 5.9±0.9 MC/mm² (P<.01) (Fig 1, left). MC density was independent of the size of thrombus (Table 1) and of the distance to the thrombus. In patients with unilateral AUTHR, tissue was obtained from the thrombosed as well as from the opposite unaffected auricle. The described augmentation and redistribution of MC were only observed in the thrombosed but not in the contralateral nonthrombotic auricle.

View larger version (12K): [in this window] [in a new window]   Figure 1. Plots show number of endomyocardial mast cells (MC) per mm2 () in thrombosed atrial appendages (AUTHR) (n=14) versus unaffected auricles (CO) (n=13). In nonthrombotic control appendages, MC were detected in the myocardium and lower endocardium, but no substantial amounts of MC were detected in the upper endocardium. In AUTHR, the number of MC increased in all endomyocardial regions, and significant numbers were detected in the upper endocardium. MC were counted as tryptase+ cells. The same numbers were obtained by counting MC in Giemsa staining.

View larger version (0K): [in this window] [in a new window]   Figure 2. Redistribution of mast cells (MC) to the upper endocardium in auricular thrombosis. A, Giemsa staining of two subendothelial MC in the presence of an atrial thrombus (THR); B, indirect immunoperoxidase staining with antitryptase of four MC in the upper endocardium (EC) located in close vicinity to the subendothelium and the thrombotic mass; C, higher magnification of two subendothelial MC in a thrombosed auricle.

Phenotype of Mast Cells in Auricular Thrombosis
Cardiac MC, both in AUTHR and in CO, were recognized by mAbs to CD9 (p24 antigen), CD29 (ß-chain of ß₁ integrins), CD33 (gp67 antigen), CD43 (leukosialin), CD44 (Pgp-1 homing receptor), CD54 (ICAM-1), CD61 (ß-chain of ß₃ integrins), ₁-antitrypsin, ₁-antichymotrypsin, and membrane-bound IgE. A negative control, the ß₂-integrin CD11b, a basophil granulocyte marker, was not expressed on endomyocardial MC. Thus, cardiac MC did not differ in their phenotype from classic MC (eg, lung MC²⁶ ). More than 90% of MC in AUTHR expressed both tryptase and chymase and were of the so-called TC-type of MC.²⁷ This phenotype corresponded to the one observed in control hearts.

Heparin could be located in MC by berberine sulfate staining. Both MC in AUTHR and MC in CO stained positive; no significant differences in the staining intensity were observed. Heparinase pretreatment of adjacent sections resulted in a markedly decreased MC labeling, whereas slides incubated with the buffer showed intense staining for MC heparin (not shown).

To evaluate the expression of c-kit (CD117), the ratio between c-kit⁺, tryptase⁺, and Giemsa⁺ MC (Fig 3) was established in serial sections (Fig 4). These data were confirmed by double-staining technique (Fig 5). Expression of c-kit was demonstrable on virtually all MC in the myocardium of thrombotic appendages (Figs 4A, 4B, 5A, and 5B) and control auricles. Identical results were obtained when different c-kit mAbs (n=6) were used. The ratio c-kit⁺:tryptase⁺ MC in the myocardium was 0.98±0.02 in control sections (Fig 3A) and 0.93±0.04 in thrombosed auricles (Fig 3B). However, in AUTHR, the redistributed MC in the upper endocardium were found to express only low or undetectable levels of c-kit (Figs 4C, 4D, 5C, and 5D). The ratio of c-kit⁺ to tryptase⁺ MC was 0.27±0.09 for upper endocardial MC in AUTHR (Fig 3D). We could not detect substantial amounts of MC in the upper endocardium of unaffected nonthrombotic appendages either with c-kit or with tryptase mAbs (Fig 3C). There were no differences between the ratio of c-kit⁺ to tryptase⁺ MC and c-kit⁺ to Giemsa⁺ MC. The ratio of tryptase⁺ to Giemsa⁺ cells was 0.98±0.05, independent of the histological region. These data show that only 18% to 36% of MC in the upper endocardium of thrombosed auricles expressed c-kit, whereas in the myocardium of both normal and thrombosed auricles, more than 90% of MC stained c-kit positive.

View larger version (26K): [in this window] [in a new window]   Figure 3. Plots show correlation between c-kit+ and tryptase+ mast cells (MC). Adjacent sections were stained either with anti–c-kit or antitryptase mAbs. , Number of c-kit+ vs tryptase+ cells in each section. In the myocardium of both normal (A) and thrombosed (B) appendages, more than 90% of MC were c-kit+ as well as tryptase+. In the upper endocardium of auricular thrombosis (AUTHR), only 27±9% tryptase+ MC were found to express c-kit (D). Only a few MC were found in the upper endocardium of some unaffected control (CO) appendages (C). Same results were obtained when correlating c-kit+ to Giemsa+ MC or counting c-kit+ vs tryptase+ cells in a double staining on the same slide. R indicates the ratio of c-kit+ to tryptase+ cells.

View larger version (0K): [in this window] [in a new window]   Figure 4. Decrease of c-kit/CD117 expression on mast cells (MC) in the upper endocardium of auricular thrombosis (AUTHR). Immunohistochemical staining with monoclonal antibodies (mAbs) to tryptase (A, C) and c-kit (B, D) was performed on adjacent sections. In the myocardium, MC were recognized by mAbs to tryptase (A) as well as c-kit (B) (x400). In the upper endocardium of AUTHR, five MC reacted with antitryptase mAb (C) but not with mAb to c-kit (D) (x100).

View larger version (0K): [in this window] [in a new window]   Figure 5. Double staining of mast cells (MC) with monoclonal antibodies (mAbs) to c-kit and tryptase. Expression of c-kit with mAb YB5.B8 was visualized by a biotinylated second-step mAb and FITC-streptavidin (A, C). Simultaneous expression of tryptase was demonstrated by use of antitryptase mAb conjugated with alkaline phosphatase and fast blue salt (B, D). A and B represent a simultaneous double labeling of three MC in the myocardium of a patient with auricular thrombosis. MC are c-kit+ (A) as well as tryptase+ (B). C and D represent the upper endocardium (EC) in the same slide. Two MC (marked by ) are tryptase+ (D) but fail to express c-kit (C).

Detection of MGF in Auricular Thrombosis
In AUTHR, a strong staining for MGF could be substantiated in the endocardium, especially in the endothelium and the subendothelial space (Fig 6B). In contrast, no reactivity could be detected in the endocardium of unaffected auricles (Fig 6A). In patients with unilateral AUTHR, the endocardium of nonthrombotic contralateral appendages did not react with mAbs to MGF. We found no differences in the staining of myocardium between AUTHR and CO. Two different anti-MGF mAbs gave identical staining patterns. Binding of both mAbs to MGF could be completely blocked by preincubation with recombinant human MGF (Genzyme).

View larger version (0K): [in this window] [in a new window]   Figure 6. Overexpression of mast cell growth factor (MGF) in the upper endocardium of auricular thrombosis (AUTHR). An intense staining for MGF was detected in the upper endocardium (EC) of appendages in AUTHR (B). No reactivity was found in the EC of unaffected nonthrombotic auricles (A). In both samples, myocardium (MYO) was reactive to anti-MGF mAbs. No augmentation of inflammatory cells was observed in AUTHR.

Detection of Leukocytes and Leukocyte Adhesion Receptors
To screen for the presence of inflammatory cells, mAbs to typical leukocyte markers such as CD3, CD45, CD68, Leu M-1, L26, myeloperoxidase, and lysozyme (Table 2) were applied. No significant differences between thrombotic, contralateral nonthrombotic, and unaffected control appendages, either in the myocardium or the endocardium, were observed. Thus, MC were the only cell type significantly increased in thrombotic atrial appendages.

Vascular endothelial cells in the myocardium of AUTHR and CO reacted as positive with anti–ICAM-1(CD54) and anti–PECAM-1(CD31) but not with anti–VCAM-1(CD106) and anti–ELAM-1(E-Selectin) mAbs. In addition, the endocardium of both thrombosed and unaffected auricles reacted as positive with anti–PECAM-1 but only weakly with anti–ICAM-1 and did not react with anti–VCAM-1 and anti–ELAM-1. Upregulation of the endothelial surface receptors ICAM-1, ELAM-1, VCAM-1, and PECAM-1 in AUTHR was not observed either in the endocardium or in the myocardium.

Histamine Release of Cardiac Mast Cells
Isolation of cardiac MC from atrial appendages was performed as described in "Methods." The percentage of isolated MC was between 2% and 5%. Nonspecific, spontaneous histamine secretion was always below 5% indicating excellent viability. Activation of cardiac MC via the c-kit receptor (CD117) by recombinant human MGF was followed by specific dose-dependent and nontoxic mediator secretion. Table 3 shows the histamine release of three different donors at various concentrations of MGF. Optimal concentrations of MGF induced 14% to 19% histamine release in human cardiac MC.

View this table: [in this window] [in a new window]   Table 3. MGF Induces Histamine Release in Human Cardiac MC

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The atrial appendage is a muscular chamber acting as a contractile pump with a characteristic pattern of contractions. However, the exact physiological function of the atrial appendage is still unknown. The reason for the predilection of the appendage in atrial thrombus formation may be not only the distinct anatomy, as its inner surface is marked by muscular ridges, but also the abnormalities in blood flow patterns. Transesophageal echocardiographical examinations in patients with atrial fibrillation revealed a significantly reduced or absent blood flow with stasis of blood.^{28 29 30} Other studies have shown that atrial appendage thrombus formation went along with auricular dilatation as well as decreased contraction.^{2 29} As clinical studies could not entirely clarify the pathogenesis of atrial thrombus formation, our group tried to examine thrombosed auricles on histological changes in comparison to unaffected atrial appendages.

We could demonstrate a significant augmentation of MC in AUTHR and their redistribution to the upper endocardium, accompanied with an overexpression of the c-kit ligand MGF. To exclude the possibility that these changes in thrombosed appendages were due to an unspecific inflammatory process, we screened for the augmentation of other inflammatory cells such as macrophages, lymphocytes, and polymorphonuclear leukocytes. However, none of these cell types was significantly increased either in the endocardium or myocardium of AUTHR if compared with contralateral unaffected appendages or control hearts, indicating that MC augmentation was an inflammation-independent phenomenon. This observation was supported by the fact that no upregulation of the leukocyte adhesion molecules ICAM-1, VCAM-1, ELAM-1, and PECAM-1 could be substantiated in AUTHR.

A possible link between MC and thrombosis has already been considered. Studies have shown that MC-deficient mice are hyperresponsive to thrombogenic stimuli.^{31 32} In 1958, Pomerance³³ observed an increase of MC in the adventitia of thrombosed coronary arteries. Adventitial MC increase was also observed in coronary thrombosis in cocaine abusers and early lesions of atherosclerosis.^{34 35}

In this report, we demonstrate an association between thrombosis of the atrial appendage and specific MC augmentation, changes in MC distribution, and local changes of MC phenotype. These phenomena were not observed in fibrillating nonthrombotic contralateral appendages of the same patients or unaffected auricles of control patients suffering from atrial fibrillation. Moreover, no significant correlation between the number of MC and the likely onset of atrial fibrillation could be substantiated. Thus, atrial fibrillation is not likely to trigger MC augmentation.

MC redistribution in AUTHR is associated with an overexpression of the MC agonist MGF in the upper endocardium. This cytokine promotes the development of human MC from their progenitors in bone marrow³⁶ and peripheral blood.¹⁸ Additionally, it is a chemotactic factor increasing the directional motility of MC.³⁷ Both effects of MGF could contribute to the MC augmentation in AUTHR. Whether the observed MC accumulation in the endocardium of thrombosed auricles is due to local differentiation from progenitor cells or due to chemoattractance of mature MC remains to be clarified.

The hypothesis of MGF as a key regulator of MC distribution is supported by several observations. Longley et al³⁸ found altered distribution of MGF with abnormal production of the ligand in cutaneous lesions of patients with mastocytosis. They speculate that an increase of MGF may cause accumulation of MC. Galli et al³⁹ observed a remarkable expansion of the MC population in many tissues and organs after administering human recombinant c-kit ligand to monkeys. Thus it is reasonable to assume that locally overexpressed MGF plays an important role in the accumulation of MC in the upper endocardium of AUTHR.

Local overexpression of MGF combined with MC augmentation prompted us to a closer examination of the c-kit receptor on MC. Redistributed MC in the upper endocardium displayed weaker or absent reactivity with mAbs to c-kit when compared with MC in the lower endocardium or myocardium of thrombosed appendages or MC in control auricles. One explanation could be that the receptors were covered by endogenous MGF, their natural ligand. On the other hand, MGF is known to cause downregulation of c-kit protein.⁴⁰ The loss of c-kit receptors from the cell surface could be explained by internalization after ligand receptor interactions.⁴¹

Recent data suggest that MGF activates MC via the c-kit receptor.^{15 17} Thus, our finding of MGF overexpression and c-kit downregulation in AUTHR might have functional implications. MC, which play an exclusive role compared with cells that are devoid of preformed granules, can release their mediators as a consequence of MGF stimulation. This has already been demonstrated for lung MC.¹⁷ Recently published data of our group²⁵ and experiments done for this study demonstrate that cardiac MC operate according to the same mechanisms. There is accumulating evidence that the c-kit signal transduction pathway can also be activated through intimate cell-to-cell contact between proliferating cells expressing the c-kit receptor and tissue-anchored stromal cells expressing the membrane-bound ligand.^{15 42} Thus, it is tempting to speculate that the overexpressed c-kit ligand in the upper endocardium may cause mediator release in locally augmented MC.

Recent studies indicate that MC can produce tumor necrosis factor- (TNF).⁴³ The hypothesis that MC-derived TNF activates endothelium and enhances thrombus formation appears unlikely considering the absence of TNF-associated upregulation of endothelial activation markers like ICAM or VCAM^{44 45} in AUTHR. On the other hand, MC are a unique source of heparin.^{6 46 47} We could demonstrate also that MC in thrombosed atrial appendages express heparin, which is well known for its anticoagulant properties. These potentially protective mechanisms of MC invite the assumption that MC redistribution to the upper endocardium in thrombosed auricles might be in order to prevent further apposition of thrombotic material and thus redress the local hemostatic balance. This concept is supported by our finding that MC accumulation and redistribution were found throughout thrombosed auricles, unrelated to the location of the thrombus. The demonstration of functional consequences, that is, mediator release of cardiac MC following MGF stimulation via the c-kit receptor, indicates a possible role for both MC and MGF in the pathology of AUTHR.

Summary
Our data clearly demonstrate an association between atrial appendage thrombosis and MC augmentation, MC redistribution, and MGF overexpression. We hypothesize that augmented and redistributed MC and their mediators may play an important role in pathophysiology of atrial thrombosis.

	Acknowledgments

 
This study was supported by FWF, grants P-10427 and P-9359, and the Bundesministerium für Wissenschaft und Forschung. We want to thank Isabella Mosberger, Sabine Muthenthaler, Karina Plesch, and Gerda Schorsch for excellent technical assistance.

Received June 17, 1994; accepted August 31, 1994.

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