Quantitative analysis of human heart valves

Cardiovascular Pathology 11 (2002) 251 – 262

Quantitative analysis of human heart valves Does anorexigen exposure produce a distinctive morphological lesion?$ Paul C. McDonalda,b,c, Janet E. Wilsona,b,c, Min Gaod, Shannon McNeilla,b,c, John J. Spinellid, O. Dale Williamse, Salima Harjia,b,c, Jennifer Kenyona,b,c, Bruce M. McManusa,b,c,* a

University of British Columbia, McDonald Research Laboratories, Room 292, 1081 Burrard Street, Vancouver, BC, Canada, V6Z1Y6 b The iCAPTUR4E Center, Department of Pathology and Laboratory Medicine, St. Paul’s Hospital, Vancouver, Canada c Providence Health Care, University of British Columbia, Vancouver, Canada d Centre for Health Evaluation and Outcomes Sciences, St. Paul’s Hospital, Vancouver, Canada e Division of Preventive Medicine, University of Alabama at Birmingham, Birmingham, AL, USA Received in revised form 28 March 2002; accepted 26 April 2002

Abstract The need for more detail regarding the clinical and morphological features of human heart valves has become evident due to recent controversy regarding anorexigen-associated valvular dysfunction. In the present study, we used quantitative digital image analysis of geometric and compositional features to compare the histopathology of cardiac valves excised from patients treated with anorexigens as compared to normal, floppy, rheumatic and carcinoid valves. Anorexigen-exposed valves had the greatest number of onlays/valve ( P < .0001), while rheumatic valves showed the greatest average onlay size and thickness of the comparison groups studied ( P = .01). The valve onlays from anorexigen-exposed, carcinoid and floppy valves contained a greater percentage of glycosaminoglycans (GAGs) as compared to normal and rheumatic valves ( P = .01). The anorexigen-exposed valve propers contained more GAGs than any other comparison group ( P = .02). Vessels were prominent in both onlay and valve proper regions of carcinoid valves, in the anorexigen-exposed valve onlays and in rheumatic valve propers. Thus, the number of onlays, their size, the degree of GAG deposition, and the presence and location of vessels and leukocytes were important features distinguishing anorexigen-exposed valves from normal valves. Discriminant analyses, based on geometry, color composition or color composition, and vessel and leukocyte counts combined, were able to separate the valves into distinguishable groups. Our findings demonstrate that specific microscopic features can be used to separate anorexigen-associated heart valve lesions from normal valves and valve lesions associated with other pathologies, and suggest that a distinctive pathological process may exist in many anorexigen-exposed valves. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Heart valve; Floppy valve disease; Rheumatic valve disease; Carcinoid valve disease; Anorexigen exposure; Fenfluramine; Phentermine; Quantitative image analysis

1. Introduction The need for greater detail regarding the clinical and morphological features of normal and diseased human heart valves has been fostered by the recent discussions and

$ Sources of support: unrestricted grant from Wyeth-Ayerst Research; P.C.M. is a recipient of a Heart and Stroke of Canada Research Traineeship. Abbreviations: fen, fenfluramine; phen, phentermine; dexfen, dexfenfluramine; SSRI, selective serotonin reuptake inhibitor; GAGs, glycosaminoglycans; VIC, valvular interstitial cell * Corresponding author. Tel.: +1-604-682-2344 ext. 62490; fax: +1604-806-8351. E-mail address: [email protected] (B.M. McManus).

controversy arising from anorexigen-associated valvular dysfunction. When the therapeutic combination of fenfluramine (fen) and phentermine (phen) was linked by investigators at the Mayo Clinic in Fargo, ND to regurgitation of both mitral and aortic valves [1], pharmaceutical companies rapidly and voluntarily withdrew fen and dexfenfluramine (dexfen) from the market. A large number of clinical studies have since been initiated in an attempt to define the existence [2– 7], nature [8 –10], severity [11 –14] and potential reversibility [15 – 21] of any valvular dysfunction (or lesion) that might arise in association with the use of anorexigens. These investigations have led to better definition of many clinical aspects, including disease prevalence, but the potentially unique histopathology of such valves has received far less attention [22].

1054-8807/02/$ – see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 1 0 5 4 - 8 8 0 7 ( 0 2 ) 0 0 11 0 - 2

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When the present study was initiated, clinical findings suggested that left-sided heart valves were affected to a greater extent by anorexigenic agents than right-sided heart valves, including evidence of mild aortic valve regurgitation [1,3,12] and a lesser frequency of mitral valve regurgitation [1]. No data from controlled studies suggested tricuspid or pulmonic valve dysfunction. Information has begun to emerge that the occurrence of valvular dysfunction is dependent upon duration of therapy and drug dosages [10,23]. Gardin et al. [10] have reported a higher prevalence of aortic regurgitation in patients exposed to fen/phen or dexfen versus controls (13.7% fen/phen, 8.9% dexfen, 4.1% untreated group), with no differences in valvular dysfunction when treatment was less than 3 months. Similarly, Jollis et al. [23] evaluated 1163 patients who had taken fen/phen and 672 control patients. Valvular abnormalities occurred primarily after patients were on the drug regimen for more than 6 months and dysfunction was characterized predominantly by mild aortic regurgitation (9% of patients as compared to 4% of normal controls, P < .001). Mild regurgitation was also evident in the mitral, tricuspid and pulmonic valves in the treated patients, but this tendency was not statistically different from controls. Importantly, valvular dysfunction associated with fen/ phen may be reversible upon cessation of the drugs [15,16,18 –20,24]. Weissman et al. [3,19] have demonstrated that aortic and mitral regurgitation were in excess in dexfen-treated patients versus controls wherein the average duration of therapy was 72 days and the median time of follow-up echocardiogram after cessation of drugs was 34 days. Recently, Gardin et al. [20] have completed a one-year follow-up on the original cohort of dexfen-treated and control subsets, and their data indicate that reversibility of dysfunction is to be expected in the vast majority of patients. Despite the work published to date, much of which relies on echocardiographic data, few studies have described the pathological changes to heart valves that result in the reported regurgitant valve dysfunction associated with exposure to anorexigens [22]. Indeed, a very limited number of preliminary and anecdotal pathological observations have been published on valves from patients taking anorexigens who, upon clinical evaluation for valvular regurgitation, underwent either aortic and/or mitral valve replacement [1,25,26], or who had right ventricular endomyocardial biopsy [27]. Most recently, a larger study was published describing the histopathology of aortic and mitral valves removed from 64 patients treated with anoretic agents [28]. The aim of the present study was to more completely define the distinguishing histopathologic features of valves taken from individuals exposed to anorexigens in the context of normal valve histology and the histopathology of other diseased valves. We have examined quantitatively the geometry and composition of mitral and aortic valves removed surgically from patients treated with anorexigens in comparison to normal valves and in comparison to those from

patients with rheumatic and floppy valve disease. In addition, we have compared compositional features from a small number of tricuspid and pulmonic valves from patients treated with anorexigens and a substantial number of right-sided heart valves affected by carcinoid valve disease to the floppy, rheumatic and anorexigen-exposed aortic and mitral valves. An effort was made through both univariate and discriminant analyses of quantitative variables to establish the distinctive microscopic features that separate one valve group from another.

2. Materials and methods 2.1. Valve identification Normal aortic and mitral valves and rheumatic, floppy and certain carcinoid valves were identified in the Cardiovascular Registry, St. Paul’s Hospital — University of British Columbia in a fashion previously described [29]. Carcinoid valves and those valves from patients exposed to anorexigens were obtained from the Cardiovascular Registry, St. Paul’s Hospital — University of British Columbia, Clarkson Hospital, Northwestern Memorial Hospital, Lehigh Valley Hospital, the Mayo Clinic, Brigham and Women’s Hospital, Stanford University and the Cleveland Clinic. A total of 270 patients ranging in age from 15 to 84 years and a total of 472 valves were included in the study (Table 1). Each valve was assessed twice grossly (when a gross valve was available) and microscopically by a cardiovascular pathologist blinded to clinical parameters and initial clinical impressions to establish the diagnosis of floppy or rheumatic disease and to exclude endocarditic valves. The number of leaflets and cusps from each study group to be compared in the analysis was not delineated a priori, since many of the fen/phen-associated and carcinoid valves received in the laboratory had already been embedded in paraffin. Thus, unlike for the normal, rheumatic and floppy valves, the number of tissue pieces, their orientation upon sectioning and the architectural completeness of sectioned leaflets or cusps of fen/phen exposed and carcinoid valves could not be controlled. 2.2. Tissue processing and staining The normal, rheumatic and floppy valves were processed as previously described [29]. As noted, all other valves were received already embedded in paraffin blocks. Three micron serial sections were cut and placed on Superfrost slides (Fisher Canada, Ottawa, ON) for histochemical and immunohistochemical staining. Serial sections of aortic, mitral, pulmonic and tricuspid valves were stained with hematoxylin and eosin (H&E) or modified Movat’s pentachrome stain using a Tissue Tek automated staining system (Sakura Finetek, Torrance, CA). Immunohistochemical staining was performed on a subset of the valves for white blood cells

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Table 1 Selected patient characteristicsa Comparison group

Normal

Rheumatic

Floppy

Anorexigen-exposed

Carcinoid

Number of patients Age range (years) Mean age (years) Sex (male, female) Height (cm)

113 15 – 84 41.2 ± 14.9 61, 52 171.23 ± 13.2 161.87 ± 7.9 79.7 ± 12.5 65.18 ± 21.5 26.9 ± 4.3 25.0 ± 8.5

77 17 – 78 50.7 ± 10.7 33, 44 172.7 ± 8.0 158.4 ± 6.7 80.4 ± 14.1 65.99 ± 17.4 26.97 ± 4.4 26.12 ± 5.7

40 20 – 79 52.5 ± 18.1 28, 12 173.2 ± 9.4 161.5 ± 6.6 74.82 ± 12.1 62.8 ± 10.8 24.93 ± 3.5 24.5 ± 2.7

15 28 – 65 46.3 ± 10.8 2, 13 Not available

25 25 – 78 58.8 ± 12.3 14, 11 178.0 ± 9.3 161.2 ± 7.0 82.13 ± 15.1 67.2 ± 20.1 25.9 ± 4.4 26.15 ± 8.9

Weight (kg) BMI (kg/m2)

Male Female Male Female Male Female

Abbreviation: BMI — body mass index. a Values are expressed as mean ± S.D. where applicable.

(leukocyte common antigen [DAKO, Mississaugua, ON]: a monoclonal antibody that recognizes CD45RB and CD45 epitopes) and vessels (Factor VIII-related antigen [DAKO]: a polyclonal antibody that recognizes endothelial cells and megakaryocytes). Antibody staining was performed on a Ventana ES automated immunostainer (Ventana, Tucson, AZ) as previously described [30,31] and the conditions were optimized with respect to antigen retrieval and antibody concentrations. In brief, after deparaffinization in xylene, the slides were rehydrated in graded ethanols (100 –70%), washed with PBS and, if necessary, subjected to antigen retrieval. Staining for Factor VIII-related antigen was performed on slides pretreated with Ventana’s protease 1 for 8 min at 37
C. Staining for leukocyte common antigen was accomplished without antigen retrieval. Factor VIII-related antigen was used at a dilution of 1:4000, while leukocyte common antigen was used at a dilution of 1:25. 2.3. Geometric analysis Normal, rheumatic and floppy heart valves embedded according to our laboratory protocol [29] were used for the geometric analysis. Again, for many valves received already embedded, particularly the majority of carcinoid and anorexigen-exposed valves, optimal embedding was not possible and valves for which a complete, full-length section could not be obtained were necessarily excluded from the geometric analysis. Thus, 310 valves from 211 patients were included in the geometric component of the study. The total number of valves studied geometrically included 186 normal valves from 105 normal patients, 62 rheumatic valves from 53 patients, 36 floppy mitral valves from 36 patients, 15 valves from 7 patients exposed to anorexigens and 11 valves from 10 patients with carcinoid heart valve disease. Each Movat’s pentachrome-stained valve assessed geometrically was submitted to systematic digital image analysis using ImagePro Plus software (Media Cybernetics, Silver Spring, MD) as previously described [29]. Measurements included valve area (total, proper and onlay), onlay characteristics (number, percent onlay area and average onlay size) and total valve and valve proper thickness (average thickness

and nine measurements along the valve leaflet or cusp). These measurements have been defined in detail in an accompanying manuscript [29] and are briefly commented upon here. The percent onlay area represents the proportion of the total valve area occupied by onlay material (onlay area/ total valve area  100) and the average size of onlays refers to the area occupied by a single onlay (onlay area/number of onlays). The thickness of the total valve and valve proper was determined at each of nine standardized sites. These measurements, extending from the base to the tip, were designated as base, base –mid 1, base – mid 2, base – mid 3, mid, mid –tip 1, mid –tip 2, mid – tip 3 and tip. Onlay thickness for each of the nine measurements was obtained by subtracting the valve proper thickness from the total valve thickness. An onlay refers to ‘‘neo-tissue’’ occurring superficial to the valvular elastic membrane or plate, toward a given surface of a valve leaflet or cusp, but deep to the surface endothelial lining. Thus, a valve onlay is created by superficial expansion of valve tissue, with the elastic membrane serving to separate the ‘‘neo-tissue’’ from the remainder of the valve. Geographical separation of onlays was used as the criterion by which multiple onlays were distinguished from a single onlay. Based on this definition, an onlay with elastic fibers underlying it was considered a single onlay. 2.4. Compositional study The compositional portion of the study included 177 patients and 242 valves. Eighty-nine normal valves from 54 patients, 66 rheumatic valves from 56 patients, 27 floppy mitral valves from 27 patients, 19 valves from 15 patients exposed to anorexigens and 41 carcinoid valves from 25 patients were analyzed for valve composition. Movat’s pentachrome-stained heart valves from recent autopsy hearts and explant hearts, and valve tissues paraffinembedded at the time of an operative procedure were analyzed with ImagePro Plus software for valve proper and onlay composition as previously described [29]. Briefly, compositional analysis of the valve proper and onlays was carried out using digital image analysis. After the valve proper and each onlay were delineated, a color segmentation

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file was applied to quantitate the percent area of the valve proper and onlay occupied by muscle-like cells, collagens, elastins and glycosaminoglycans (GAGs) (Fig. 1a– e). A particular region of tissue that could not be specifically distinguished was labeled as ‘‘admixed.’’ This latter tissue, a small component of any given area assessed, is a mixture of some or all components and could not be distinguished as separate components because of the low power of magnification required in order to digitize the valve images. The Movat’s pentachrome stain does not allow accurate and quantitative delineation of two important features of heart valves, inflammatory cells and blood vessels. Thus, a subset of valves used in the compositional analysis was also assessed by immunohistochemistry to establish the number of white blood cells and blood vessels present. The patients and valve numbers in this subset included 34 valves from 23 normal patients, 22 rheumatic valves from 19 patients, 17 floppy mitral valves, 18 valves from 13 patients exposed to anorexigens and 27 carcinoid heart valves from 19 patients, for a total of 118 valves from 91 patients. White blood cells or vessels stained positively by immunohistochemistry were counted under a light microscope in the valve proper and each onlay. Thus, with ImagePro Plus area measurements, a final index of numbers of cells or vessels/ mm2 of tissue was obtained.

2.5. Statistical analysis All statistical analyses were carried out using SAS (SAS Institute, Cary, NC) on a UNIX platform. Descriptive statistics were prepared using data for all valve types and diagnoses, including means and standard deviations. It is noteworthy that basic statistical derivations such as sample means, standard deviations and t test-derived P values are not valid when making comparisons among groups where each group contains a varying set of complex variables. Thus, a mixed effect model approach, which takes into account the multiple valves per person, was used for making comparisons among groups. Variables included in these models, in addition to those designating the groups, were age, sex, diagnosis and valve type (anterior mitral valve leaflet, posterior mitral valve leaflet and aortic valve). Adjusted means were calculated from these models and Tukey – Kramer corrections for multiple comparisons were made to obtain adjusted P values. These analyses were performed for the geometric features using only aortic and mitral valves from the normal, rheumatic, floppy and anorexigen-exposed valve groups. Compositional features were assessed with this analytical approach for all valves in all groups. Stepwise discriminant analyses were used in an effort to identify a set of variables that best reveals the differ-

Fig. 1. Digital photomicrographs (6  ) of mitral valves from each comparison group. The color mask from the ImagePro Plus color segmentation file is illustrated for each valve. Color code: black: elastins, red: muscle-like cells, yellow: collagens, cyan blue: GAGs, green: admixed. Photomicrographs in Panels a, b, c and e are taken from the appropriate boxed areas in Fig. 3. (a) Normal mitral valve. Digital photomicrograph of a normal mitral valve with the color mask illustrating a range from yellow to sea-green due to the combination of collagens and GAGs. (b) Rheumatic mitral valve. Digital photomicrograph of a rheumatic valve illustrating the prominent thickening of the atrialis (right) and complex onlay material on the ventricularis (left). The color mask illustrates a mixture of collagens, elastin and mesenchymal cells (myofibroblasts and smooth muscle cells). In addition, there is prominent vascularity within the core of the valve running along the most elastotic region and within the spongiosa. These vessels may be small arteries, veins or lymphatics. (c) Floppy mitral valve. Digital photomicrograph of floppy mitral valve illustrating expansion of the atrialis (right). The color mask delineates the sea-green coloration due to prominent GAGs mixed with elastin and collagen. (d) Anorexigen-exposed mitral valve. Digital photomicrograph of mitral valve previously exposed to anorexigens. The ventricularis has a variable sea-green to partially elastin-containing appearance. Small onlays are present and are primarily GAG-rich. The color mask highlights the areas of rather prominent GAGs. (e) Carcinoid tricuspid valve. Digital photomicrograph of portions of a tricuspid valve affected by carcinoid disease illustrating prominent ventricular and atrial onlays. These onlays are quite profound. The color mask illustrates the heavily sea-green color reflecting the GAGs, as well as a rich complement of mesenchymal cells. The elastotic atrialis membrane is evident.

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ences among the diagnostic groups. Discriminant functions were derived as linear combinations of the variables that maximize the distance between groups. The values of the estimated discriminant functions for each valve, called the discriminant scores, were plotted to show the clustering of valves according to their diagnostic groups based on the identified set of variables. Discriminant analysis was carried out for the combined geometric and compositional features of the anterior mitral valve leaflet and separately for the geometric and compositional features for mitral and for aortic valves, respectively. It was also performed using data from leukocyte and vessel counts in combination with the compositional features of mitral and aortic valves, respectively. Due to limited sample data, right-sided valves were excluded from the discriminant analysis. To conserve space, the discriminant functions have not been provided, but they are available upon request from the authors.

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2.6. Study limitations Several limitations and caveats pertain to this study. First, while this study includes a sizeable number of valves (472 valves from 270 patients), the process by which such valves become available necessarily does not permit fully comfortable inferences to broader populations. Further, the nature of possible selection biases is difficult or impossible to fully answer. There is some comfort, however, in the logical pattern and the consistency of the results we report. These results do depict a fairly strong relationship within this specific data set. It is yet to be seen whether these or similar relationships can be replicated in other data sets. Second, only limited clinical data was available on these patients. Third, morphometric measures were obtained in formalin-fixed, dehydrated tissue sections so the dimensions obtained in all valves underestimate by  10% those that would exist in vivo. Fourth, the sectioning and embedding

Fig. 2. The means adjusted for age, sex and valve types from mixed effect model analyses of geometric features for cases with mitral and aortic valves. (a) Total valve area. No statistically significant differences were found among the valve groups. (b) Valve proper area. No statistically significant differences were found among the valve groups. (c) Onlay area. The rheumatic valve onlay areas were significantly greater than those in other comparison categories (A vs. R, P = .003; F vs. R, P = .01; N vs. R, P < .0001). The floppy valve onlay areas were also significantly larger than in the normals ( P < .0001). Variability in onlay areas for the anorexigen-exposed valves mitigated against statistical significance relative to normal valves. (d) Percent onlay area. As a percentage of the total tissue cross-sectional area, rheumatic valves exhibited the greatest onlay area of the valve groups (F vs. R, P = .002; A vs. R, P = .004; N vs. R, P < .0001). Floppy and anorexigen-exposed valves also had a greater percent onlay area than normal valves (F vs. N, P < .0001; A vs. N, P = .0002). (e) Average size of onlays. Onlay size on rheumatic valves was dramatically greater than those on normal valves (N vs. R, P < .0001) and larger than onlays found on either floppy valves (F vs. R, P = .005) or on anorexigen-exposed valves (A vs. R, P = .004). In addition, onlay size on floppy valves was greater than that on normal valves (N vs. F, P = .01). (f ) Number of onlays. All valve groups had more onlays when compared to normal valves (A vs. N, P < .0001; F vs. N, P < .0001; R vs. N, P < .0001). In addition, anorexigen-exposed valves had significantly more onlays than did rheumatic valves (A vs. R, P = .0008). (g) Total valve thickness. All comparison valve groups had thicker valves than normal (A vs. N, P = .0005; F vs. N, P < .0001; R vs. N, P < .0001). Rheumatic valves appear to be the thickest. (h) Valve proper thickness. Both rheumatic and floppy valves had a statistically thicker valve proper than normals (F vs. N, P = .0001; R vs. N, P < .0001).

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protocol was not possible for most anorexigen-exposed and carcinoid heart valves. Choosing a specific site for sectioning the normal, rheumatic, and floppy valves, in theory, may alter findings in a manner not known. Finally, this analysis is a cautious, but reasonable, comparison of the mitral, aortic, tricuspid and pulmonic valves, allowing that certain diseases are valve specific, including floppy mitral valves and right-sided carcinoid heart valve disease.

3. Results 3.1. Geometry In Fig. 2, the group means adjusted for age, sex and valve type from the mixed effect model analyses are presented for

those cases with aortic and mitral valves. The cross-sectional area of the total valve and the valve proper regions were not statistically different among comparison groups (Fig. 2a and b). Differences in the average valve onlay area among the groups were, however, highly significant ( P < .001). Specifically, rheumatic valves had a larger average onlay area than any of the other valve categories, while floppy valve onlay areas were larger than those of normal valves (Figs. 2c and 3a – c, f and g). When comparing the percent onlay area, we found a similar trend (Fig. 2d), with rheumatic valves having the greatest percent onlay area (35%), followed by floppy valves (25%), anorexigenexposed valves (23%) and normal valves (6%). Rheumatic valves had the greatest average onlay size (7 mm2) (Fig. 2e), followed by floppy valves (4 mm2), anorexigen-exposed valves (2 mm2) and normal valves (1 mm2). The number of

Fig. 3. Digitally scanned images of illustrative whole cross-sections of normal (a and f ), rheumatic (b and g), floppy (c), anorexigen-exposed (d and h) and carcinoid (e and i) valves. Geometric evaluation delineated small and infrequent onlays in the normal valves, the large onlays in the rheumatic (arrows, b), moderate-sized onlays in the floppy valves (arrows, c) and several small onlays in the anorexigen-exposed valves (arrows, d). Carcinoid heart valves had dramatic large onlays (arrows, e and i). The boxes delineate areas illustrated in Figs. 1 and 5.

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onlays (Fig. 2f ) was the greatest in the anorexigen-exposed valves, with approximately 3 onlays/valve (Fig. 3d and h), while normal valves had the fewest (  1 onlay/valve). Fig. 2g and h depict the average thickness of the total valve and valve proper, respectively. The average thickness of the rheumatic, floppy and anorexigen-exposed valves was found to be greater than the normal valves, with the rheumatic valves being the thickest. 3.2. Composition We recently carried out a compositional analysis of human normal heart valves and their onlays as they age using a digital imaging system [29]. We have now extended that analysis to the assessment of valves in different comparison groups. The valve proper and valve onlays were analyzed separately for their composition. Adjusted means are shown in Fig. 4 for both the valve proper and onlays, including each constituent distinguishable by digital image analysis, for the mitral or tricuspid and aortic or pulmonic valves. The mean compositional contributions to the valve onlays (Fig. 4a and c) and the valve proper (Fig. 4b and d) are illustrated to show the similarities and differences among comparison groups. In the valve proper, it can be seen that the anorexigenexposed, carcinoid and floppy valves have a greater relative amount of GAGs than the normal and rheumatic valves, while the rheumatic and normal valves have a greater percentage of collagen than the other valve cat-

Fig. 4. Histographic representations of tissue constituent contributions to the overall makeup of onlays (a and c) and the valve proper (b and d) of mitral, tricuspid, aortic or pulmonic valves relevant to particular comparison valve groups are presented. The distinctive nature of onlays and the valve proper for each comparison valve group is evident. Thus, for example, prominent differences in the contribution of collagens versus glycosaminoglycans, both within the onlays and the valve proper is appreciated between the different valve groups. Overall, the most collagenous valves are the rheumatics and the most GAG-rich valves are the anorexigen-exposed and carcinoid valves.

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Table 2 Adjusted means from mixed effect model analyses for percent of valvular leukocytes, defined as LCA-positive cells, and vessels as determined by immunohistochemical staining in the valve proper and valve onlays of normal, rheumatic, floppy, anorexigen-exposed and carcinoid valves Leukocytesa

Vesselsa

Valve group

Onlay

Proper

Onlay

Proper

Normal Rheumatic Floppy Anorexigen Carcinoid

7.9 28.7 21.1 88.2 21.5

5 4 3 4 16.8

0 0 0 3.8 15

0 5 0 1.5 12.5

a Data are expressed as number of cells or vessels per area of tissue (mm2).

egories. Features seen in the valve onlays of different valve categories are in approximate concordance with findings in the respective valve proper. In addition to alterations in basic tissue composition, the presence or absence of blood vessels and inflammatory cells can be associated with important events during certain valvular disease processes [32 – 35]. We found vessels to be frequently prominent in both onlay and valve proper regions of carcinoid valves (Table 2) and, with the exception of the valve proper of rheumatic valves, were found with statistically greater frequency than in any of the other valve groups (onlay: C vs. N, P = .008; C vs. R, P = .002; C vs. F, P = .002; C vs. A, P = .03; proper: C vs. N, P = .01; C vs. F, P = .02; C vs. A, P = .01). Importantly, the geographical distribution of blood vessels and inflammatory cells was also distinctive for certain valve categories (Fig. 5). The vessels of rheumatic valves were located mostly in the spongiosa of the valve proper (Fig. 5c), while vessels with surrounding inflammatory cells were more widespread and superficial in both the valve proper and onlays of carcinoid (Fig. 5d –f ) and anorexigen-exposed valves (Fig. 5a and b). Floppy and normal valves rarely had vessels in either the onlays or valve proper. Although leukocytes were present to varying degrees in the valve onlays of all comparison groups, they were typically more prominent in the carcinoid and anorexigen-exposed valves in both the valve proper and onlay regions (Table 2; Fig. 5a and d). Substantial numbers of inflammatory cells were also evident in the onlays of rheumatic valves (Table 2). The variability in number for each case prevented statistical significance from being reached. Overall, the anorexigen-exposed valves exhibited the greatest range of any of the comparison groups for inflammatory cells and vessels in the valve proper or the valve onlays (Table 2). 3.3. Discriminant analysis Discriminant analysis uses linear combinations of independent variables, termed linear discriminant functions, to classify cases into one of the comparator groups. The values of these estimated functions for each valve are the discrim-

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Fig. 5. Digital photomicrographs of representative valve sections immunohistochemically stained with antibodies against leukocyte common antigen for leukocytes (a and d) and Factor VIII-related antigen for vessels (b, c, e, f ). Brown coloration is indicative of positive immunoreactivity (arrows). Photomicrographs are taken from the boxed areas in Fig. 3b, e and h. Numerous leukocytes are evident in the onlays of anorexigen-exposed mitral valves (a) and carcinoid tricuspid valves (d). Vessels are prominent in the onlays of anorexigen-exposed (b) and the carcinoid (e) valves, in the same regions as the leukocytes (a and d). Large vessels are prominent in the valve proper of the rheumatic valve (c) and are present in the valve proper of the carcinoid (f ) valves. Scale bar = 50 mm.

inant scores and these scores can be plotted to describe the separation between groups. Discriminant analysis was carried out first for the three valve diagnoses, normal, floppy and rheumatic. The geometric and compositional data were combined for the anterior mitral valve leaflets and are illustrated in Fig. 6. Effective discrimination among the three groups was obtained using the variables identified in the discriminant function. Discriminant analyses were then carried out separately for the geometric data and compositional data for the mitral and aortic valves. Floppy mitral valves could not be distinguished using the discriminant analysis that included normal, rheumatic, floppy and anorexigen-exposed valves. Thus, the results for floppy valves based on the latter analysis are not presented in this paper. Discriminant analysis based on geometry of normal, rheumatic and anorexigen-exposed anterior mitral valve leaflets (Fig. 7a) included six variables. These variables were, in order of importance, onlay area, percent onlay area, base – mid 3 thickness, mid – tip 1 thickness, mid – tip 2

thickness and the tip thickness. Variables for the aortic valves, in order of importance, included percent onlay area,

Fig. 6. Discriminant analysis of the anterior mitral valve leaflets of normal (N), rheumatic (R) and floppy (F) valves. These three known clinical diagnoses are discriminated using the linear discriminant function. The solid diamonds (^) indicate the position of the centroid for each diagnostic group.

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Discriminant analysis was also performed for the subset of valves that had both color composition based on the Movat’s pentachrome staining and leukocyte and vessel counts (Fig. 7e and f). The two most discriminating features for the mitral valve were the percent area of GAGs and the number of vessels in the valve proper, while the three discriminant features for the aortic valve were the percent GAGs in the valve proper and the percent muscle-like cells and number of vessels in the valve onlays. Separation of the valves was even more distinct than when color composition alone was used.

4. Discussion

Fig. 7. Discriminant analysis of mitral valves (a, c and e) and aortic valves (b, d and f ). Valves are differentiated into three separate groups using a linear discriminant function (normal [N], rheumatic [R] and anorexigenexposed [A]). The solid diamonds (^) indicate the position of the centroid for each group. The floppy valves could not be distinguished using this analysis (data not shown). Analysis of geometric characteristics was performed for the anterior mitral valve leaflet (a) and the aortic valve (b). Separation was achieved using six variables for the mitral valve and seven similar variables for the aortic valve. Analysis of color composition was performed for the mitral valve (c) and the aortic valve (d), using four variables in each case. Discriminant analysis of color composition combined with leukocyte and vessel counts per tissue area was performed for the mitral valve (e) and the aortic valve (f ). Use of two and three discriminating variables, respectively, resulted in prominent separation of the three valve groups. The precise variables used in each analysis and a detailed description of discriminant analysis can be found in the text.

mid – tip 3 valve proper thickness, base –mid 2 valve proper thickness, onlay area, mid valve thickness, mid – tip 1 thickness and onlay number (Fig. 7b). Thus, for both the mitral and aortic valve, the most important geometric features included the size of onlay and valve thickness toward the free margin. Using this statistical model, separation of the valves into three distinct groups was achieved, particularly for the aortic valve. In a similar fashion, discriminant analysis for color composition included several discriminating variables for each valve (Fig. 7c and d). In the case of the mitral valve, these variables were percent GAGs in the valve proper, percent GAGs in the valve onlays, percent elastin in the valve onlays and percent muscle-like cells in the valve proper. The discriminant aortic valve variables were only slightly different and included the percent GAGs in the valve proper, the percent elastin in the valve proper, the percent muscle-like cells in the valve onlays and the percent collagen in the valve proper. Again, reasonable separation of the valves into three groups was achieved, especially for the aortic valve. Of particular interest, the percent of GAGs present in the valve proper was a prominent distinguishing feature for both the aortic and mitral valves.

In the first report on the echocardiographic and pathologic findings of heart valves with fen/phen exposure in obese patients [1], echocardiography revealed the presence of both right-sided and left-sided abnormalities in 24 female patients. The issue of cardiac valvular dysfunction in association with anorexigen exposure has since been the subject of several clinical investigations [3,10,16,19]. Many of these studies have relied heavily upon echocardiography [10,14,21,23], a technique that detects primarily physiological events and not necessarily fine structural features that are potentially pathologically important. In contrast, few reports exist describing the gross or histologic pathology of such functionally regurgitant valves. Right- and left-sided valves removed surgically from three patients in the original study by Connolly et al. [1] revealed histopathologic features said to be similar to those seen in ergotamine-induced or carcinoid heart disease, namely thickened, glistening leaflets as well as an appearance of shortened chordae tendineae, with matrix-rich expansion of the valve tissue. ‘‘Myxomatous’’ onlay formation on the valves of patients treated with fen/phen or dexfen was interpreted as a prominent histopathologic finding in these patients. Only two full-length reports have provided any details about the valve lesions associated with anorexigen use. Based on assessment of a small number of surgically excised valves (two mitral and one aortic), the first of these studies described focal thickening associated with increased valvular opacity as major gross features [26]. Microscopically, superficial ‘‘fibromyxoid’’ tissue was observed, as was the presence of CD3-positive leukocytes. The second, larger study described a ‘‘typical plaque’’ as containing a myxoid stroma, proliferative myofibroblastic cells and often small vessels and lymphocytic accumulations [28]. This study also highlighted dramatic interpatient variability in the severity of lesions associated with anorexigen use. While these descriptive investigations have acknowledged the presence of myxomatous and inflammatory lesions exhibiting histopathologic features similar to other valve pathologies, they have not made direct, quantitative comparisons between valve lesions associated with anorexigen exposure and normal or diseased valves.

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The current study was undertaken in an effort to establish the presence of distinctive microscopic features that separate human heart valves exposed to anorexigens from normal valves and valves exhibiting rheumatic, floppy and carcinoid lesions. It is the first attempt at discrimination of comparison valve groups on the basis of fine compositional and geometric characteristics at the histopathological level. It is important to note that several changes occur during the natural aging process of heart valves [36 –38], making the separation of lesions brought on by normal aging from those associated with anorexigen use necessary. Our recent quantitative assessment of normal valves has demonstrated that they are typically thin, with very few but definite small onlays [29]. With increasing age, normal valves become thicker, more onlays appear and the percentage of total valve area attributable to onlays becomes greater [29]. Normal valves are composed of roughly equal amounts of collagens and GAGs, and rarely harbour leukocytes and vessels, even as they age. These structural changes affect the entire valve and are most likely the result of hemodynamic, rheologic and mechanical factors operative in the valve leaflets and cusps over time. The anorexigen-exposed valves exhibited great variability, ranging from near normal in appearance to markedly abnormal. Still, we found several quantitative features that effectively separated such valves from their normal counterparts. Specifically, valves taken from individuals exposed to anorexigens exhibited a greater percent onlay area, more onlays per valve, a greater total valve thickness, a higher percentage of GAGs and a lower percentage of collagen. Numerous vessels and variable numbers of leukocytes were distributed within the valve proper and valve onlays of anorexigen-exposed valves, while these entities were rarely present in normal valves. Thus, valve thickness, valve onlay number, GAG content, vascularity and inflammation serve to separate normal and aging valves from those exposed to anorexigens. Similar features were also important in distinguishing anorexigen-exposed valves from rheumatic valves. Previous observational studies have described leaflets or cusps of valves affected by rheumatic disease as fibrotic and collagenous with associated neovessels in the valve core and inflammatory cell infiltrates consisting primarily of lymphocytes and macrophages [39,40]. In the present study, rheumatic heart valves were found to be the thickest of the valve groups analyzed, a characteristic that was particularly evident toward the valve tip. These valves had the greatest crosssectional valve area, onlay area, percent onlay area and onlay size, but yielded the smallest number of onlays per valve. The composition of the valve proper and onlays was similar, consisting primarily of collagen, a few GAGs and some smooth muscle cells and myofibroblasts. The collagen-rich nature of these valves was not unexpected, given that collagen is the characteristic matrix component associated with late healing events and valvular lesions seen in rheumatic disease by the time of valve excision would

typically be considered well-healed. These valves also harboured numerous vessels in the valve proper. In comparison to rheumatic valves, valves associated with anorexigen exposure had smaller onlay areas, percent onlay areas and average onlay sizes, greater numbers of onlays, more GAGs and less collagen. Furthermore, vessels and leukocytes, while present in both valve categories, were differentially distributed. Vessels in rheumatic valves were numerous, but were limited to the spongiosa in the valve core, while vascular structures in anorexigenexposed valves were superficial and widespread. The greater onlay number, the smaller onlay size and the GAG-rich nature of valves associated with anorexigen exposure as compared to rheumatic valves may reflect lesion age, with the small, numerous, provisional matrixrich lesions in anorexigen-exposed valves representing young, succulent lesions. More likely, these differences may signal the presence of a distinct pathogenetic process in anorexigen-exposed valves. Published qualitative gross and microscopic features of floppy mitral valve leaflets include lengthening of leaflets and chordae, interchordal hooding, increased GAGs (myxomatous transformation) and thickening of the spongiosa [41]. In our study, the thickness, average onlay area and percent onlay area of floppy valves were intermediate between that seen in normal and rheumatic valves, while the number of onlays per valve was greater in floppy as compared to rheumatic valves. In contrast to the collagenrich rheumatic valves, floppy valves were composed of roughly equal amounts of collagen and GAGs. This tissue composition was reflected in both the valve proper and the valve onlays, although a trend toward increased GAGs in the valve proper was noted. Distinctive microscopic features separating anorexigen-exposed valves from floppy valves were limited to valve composition and vascularity, as none of the geometric features outlined in this study were significantly different between the two valve groups. Valves exposed to anorexigens were more GAG-rich than floppy valves, both in the valve onlay and the valve proper. In addition, while blood vessels were generally absent from floppy valves, they were a prominent feature of anorexigenexposed valves. Thus, while both floppy mitral valves and anorexigen-exposed valves are considered to have undergone myxomatous degeneration, the greater proportion of GAGs and the presence of vascular structures in anorexigen-exposed valves aids in separating these two valve pathologies. Furthermore, the differences suggest that the pathobiological mechanisms involved in the generation of valve lesions associated with anorexigen use may be, in part, different from those operant in floppy mitral valves. Valvular lesions associated with carcinoid disease have been described as glistening, superficial ‘‘plaque-like’’ thickenings composed of GAG-rich tissue on the surface of valvular leaflets and cusps [35,42,43]. Such lesions have been reported to be similar to the myxomatous valve lesions associated with anorexigen use. In our study, carcinoid

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valves were evaluated based on composition alone. The valve proper of the carcinoid valves contained approximately equal proportions of GAGs and collagen, while the onlays of these valves were significantly GAG-rich. Both the valve proper and onlays of the carcinoid valves had a large number of leukocytes and vessels per square millimeter of tissue area. In comparison to valves affected by carcinoid disease, valves associated with anorexigen exposure exhibited even greater GAG deposition in the valve proper, while significantly fewer vessels were present. Such features indicate the presence of a particularly myxomatous process within the valve itself and further suggest the existence of a distinctive pathologic process in valves associated with anorexigen exposure. Recognizing that a distinctive pathological process may be involved in the lesions of anorexigen-exposed valves, we sought to validate our data demonstrating differences in geometry and composition among valve groups using an independent statistical approach. Discriminant function analysis provided a statistical tool that enabled us to separate the valves into distinguishable groups. Discriminant function analyses were prepared for the aortic and mitral valve separately, using the quantitative measures of tissue geometry and composition discussed above. The presence of onlays on the valves and the size of such onlays represented important geometric features that could be used for discrimination. GAGs as a percent of tissue area was the most important discriminating factor for analyses using either color composition alone or color composition in concert with leukocyte and vessel counts. The presence of vessels also had a prominent influence on discriminating valve groups. The majority of mitral valves from patients exposed to anorexigens were distinguishable from the other valve groups based on geometry and composition using discriminant function analysis. Two anorexigen-exposed mitral valves were, however, visibly different in histologic makeup when compared to all other anorexigen-exposed valves. In fact, these two valves could not be distinguished from rheumatic valve disease on the Movat’s pentachrome stain when discriminated using color composition alone or color composition in combination with vessels and leukocytes. The inability to separate these two valves may be related to the presence of pre-existent valve disease, although constraints on the acquisition of clinical information necessarily requires that such a conclusion remains speculative.These two valves also highlight the diagnostic dilemma involved in separating some valves exposed to anorexigens from valves with post-rheumatic scarring and demonstrate the importance of defining distinctive, quantitative features that differentiate anorexigen-associated valvulopathy from other valve pathologies. In conclusion, we have demonstrated distinctive microscopic features that separate human heart valve lesions associated with anorexigen exposure from normal valves and valve lesions associated with other pathologies. The

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number of valve onlays, their size, the degree of GAG deposition and the presence and location of vessels and leukocytes were important features distinguishing anorexigen-exposed valves from normal and rheumatic valves, and enabled their separation by discriminant function analysis. While discriminant function analysis was unable to clearly separate anorexigen-exposed valves from floppy mitral valves in this study, greater deposition of GAGs and the presence of vessels and leukocytes in the former valve group aid in distinguishing between these myxomatous pathologies. Further, while certain of these features are reminiscent of those seen in carcinoid valves, the phenotype observed in the anorexigen-exposed valves available for analysis was less dramatic and more variable from the point of view of average onlay size and insofar as the extent of inflammation and vascularity. The presence of a myxomatous process in both the valve core and the onlay suggests the presence of pathogenetic mechanisms distinct from those involved in floppy mitral valve disease and carcinoid disease. The range of features found in the anorexigenexposed valves no doubt reflects the duration of exposure, the drug dosages and individual patient susceptibility factors, including local standards and patterns of care and the influence of pre-existent valve disease. Unfortunately, because of constraint on the acquisition of clinical information, ascertainment of the number of patients with preanorexigen valvular dysfunction could not be pursued. Finally, the provisional matrix-rich, inflamed and neovascularized nature of the onlays and of the valve proper of anorexigen-exposed valves suggest the opportunity for regressive structural and functional changes following discontinuation of the drugs [15,16,24].

Acknowledgments The authors would like to thank Salima Harji, Sylvia Loo and Albert Lee for their assistance in sectioning the valves and obtaining patient information; Stuart Greene and Joe Comeau for assistance in setting up the computer-based imaging; Amrit Mahil and Margaret McLean for their excellent histological skills; and Heidi Wiebe, Laura Kuyper, Jill Robson and Jennifer Kelly for technical assistance. The authors would also like to thank Drs. Gerald Berry, Robert Bonow, Bill Edwards, William Laskin, Stanley Radio, Norman Ratliff, Gayle Winters and Robert Yowell for provision of valvular tissue for these studies.

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