Morphological Changes In Small Vessels On Endomyocardial Biopsy

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1 ANNALS O F CLINICAL AND LABORATORY SCIEN C E, Vol. 16, No. 3 Copyright 1986, Institute for Clinical Science, Inc. Morphological Changes In Small Vessels On Endomyocardial Biopsy SHI-KAUNG PENG, M.D., Ph.D. and WILLIAM J. FRENCH, M.D. Departments o f Pathology and Medicine, Harbor-UCLA Medical Center, Torrance, CA ABSTRACT Small vessel disease has been described in various cardiac conditions including diabetes mellitus, amyloidosis, and connective tissue disease. Less well understood is the incidence and morphological features of small vessel disease in patients with myocardial disease of unknown etiology. This study examines the incidence, clinical presentation, and pathological changes of small vessel disease in patients with normal epicardial coronary arteries undergoing endomyocardial biopsy. Biopsy specimens in 110 consecutive patients w ere analyzed by light and electron microscopy. Small vessel abnormalities w ere present in 16 patients (14.6 percent) of whom five patients had associated hypertension and 11 patients had idiopathic small vessel disease. There w ere six males and 10 females with a mean age of 53 (26 to 76) years. Clinical presentations were arrhythmias, heart failure, or chest pain. The left ventricular ejection fraction was reduced (<50 percent) in 12 of these 16 patients. The morphological features of small vessel disease included m arked thickening of the arterial wall owing to subendothelial deposits of heterogenous electron dense materials consisting of microfibrils, collagen and elastic fibers, cellular debris, and other amorphous substances. Subendothelial deposits comprised a mean 60 percent (40 to 76 percent) of the arterial wall thickness. Introduction Large epicardial coronary arteries are th e m ost com m on site of pathological involvem ent, particularly from atherosclerosis. Sm all coronary a rte rie s or arterioles also may becom e obstructed in patients with diabetes mellitus, amyloidosis, connective tissue diseases, and other pathological conditions which are less well understood.7 The incidence and clinical presentation of patients w ith sm all v e sse l d ise a se a re u n k n o w n. Obstruction of small vessels may im pede coronary blood flow, and th e clinical presentation would be indistinguishable from that of obstruction in large epicardial coronary arte rie s. To assess the pathological changes in small coronary vessels, endom yocardial biopsy specim ens from 110 consecutive p atien ts w ere stu d ie d by lig h t an d e le c tro n microscopy and analyzed by m orphom etric methods /86/ $01.50 Institute for Clinical Science, Inc.

2 Materials and Methods C l in ic a l E v a l u a t io n There w ere 110 consecutive patients included in this study, 64 males and 46 females with a m ean age of 52 years and ranging from 14 to 76 years. The medical reco rd of each p a tie n t was review ed retrospectively. The most common indication for endom yocardial biopsy was suspected inflam m atory myocarditis in patients with a recent onset (<one year) of unexplained heart failure which was usually associated w ith a reduced left ventricular ejection fraction. Other indications included chest pain, a rrh y th mias, sudden death, and assessment of doxorubicin cardiotoxicity and cardiac allograft rejection. All patients had normal epicardial coronary arteries by coronary arteriography. E n d o m y o c a r d ia l B io p s y Cardiac catheterization was perform ed in all patients using standard techniques. At the conclusion of the procedure, multiple biopsy specimens w ere taken from the right ventricular septum. T hree to six sp ecim en s of b io p sy tissu e w ere obtained in each patient. The specimens m easured one to two m m in size and were im m ediately im m ersed in Bouin s solution for light microscopy and in 2.5 percent buffered glutaraldehyde solution for electron microscopy. In select cases, d irect im m u n o flu o rescen ce was also studied. Pa t h o l o g ic E v a l u a t io n PATHOLOGY OF SMALL CORONARY VESSELS 181 All histological sections w ere stained w ith hem otoxylin and eosin, M asson trichrome, periodic acid-schiff, and elastic van Gieson s stains. In selected cases specimens w ere stained with Congo red and Prussian blue. Electron microscopic (EM) specimens were em bedded in plastic resin. Thick sections of the EM specimens w ere stained with toluidine blue. Thin sections w ere taken from selected areas, stained w ith uranyl acetate and lead citrate, and viewed under a Hitachi electron microscope. In addition to ro u tin e evaluation of endocard iu m, m yocardium, in te rstitium, and intram ural blood vessels, the diam eter of myocardial cells and extent of in terstitial fibrosis w ere m easured morphom etrically using Micro-plan II.* S p ecific sco res w e re g iv en to each biopsy. M yocellular hypertro p h y was classified as 0 or absent (<15 (Jim ), 1 or mild (15 to 20 p-m), 2 or m oderate (20 to 25 xm) and 3 or severe (> 25 pim). Interstitial fibrosis was also scored as 0 or absent (< 5 percent), 1 or mild (5 to 10 percent), 2 or m oderate (10 to 20 percent) and 3 or severe (>20 percent). Myofibrillar degeneration was e v a lu a te d by e le c tro n m icroscopy. Although small vessel changes can be seen in light m icroscopic exam ination, the degree of involvement may only be evident on electron microscopic examination. The thicknss of the subendothelial zone as com pared to the total vessel wall thickness also was m easured m orphometrically. Results Pa t h o l o g ic a l C l a s s if ic a t io n o f E n d o m y o c a r d ia l S p e c im e n s Cardiomyopathy: Biopsy specimens w ith m yocellular h y pertrophy greater than 15 (xm, interstitial fibrosis of more th a n five p e rc e n t, and m y o fib rilla r degen eratio n on electron m icroscopy w ere considered to be consistent with a cardiomyopathic process. Myocarditis: Active myocarditis was diagnosed on th e basis of m yocyte * L aboratory C om puter System, Inc. C am bridge, MA.

3 182 PENG AND FRENCH necrosis and inflam m atory cell aggre gates or lymphocytic infiltrates greater than five lymphocytes per high power field in 20 randomly selected areas. To minimize sampling error, a minimum of three samples were obtained for analysis from each patient. Amyloidosis: Congo red stain of the biopsy specimens showed apple-green b irefrin g en t m aterials, and electron microscopy showed the presence of amy loid fibrils. Interstitial fibrosis: Biopsy speci mens showed only interstitial fibrosis without myocellular hypertrophy or con spicuous myofibrillar degeneration. Small vessel disease: In tram u ral small arteries or arterioles (20 to 100 jlin in diameter) showed marked wall thick ening owing to widening of subendothelial zone and/or medial hypertrophy (fig ures 1 and 2). The subendothelial zone contained heterogenous electron dense materials, such as microfibrils, collagen and elastic fibers, cellular debris, or other amorphous substances (figures 3 and 4). M orphometric measurements of subendothelial deposits revealed that FIGURE 1. I n t r a m y o c a r d ia l s m a ll a r te r io le s h o w in g w id e n in g o f s u b e n d o th e lia l z o n e a n d p e riv a s c u la r fib ro s is. (x 600)

4 PATHOLOGY OF SMALL CORONARY VESSELS 183 FIGURE 2. I n tr a m y o c a r d ia l s m a ll a r te r y s h o w in g m a r k e d th ic k e n in g o f v e s s e l w a ll w ith w id e n in g o f s u b e n d o th e lia l z o n e a n d m e d ia l h y p e r tro p h y. ( x 30 0) F ig u r e 3. U l t r a s t r u c t u r e o f a r t e r i o l e s h o w in g m a rk e d w id e n in g of s u b e n d o th e lia l zo n e ( > «) c o m p o s e d o f h e t e r o g e n o u s e le c tro n d e n se m a te r ia l, 8,0 0 0 ) (x

5 184 PENG AND FRENCH F i g u r e 4. H ig h e r m a g n if ic a tio n o f s u b e n d o th e lia l z o n e s h o w in g m ic ro fib rils, f r a g m e n te d e la s tin, a m o r p h o u s g r a n u la r s u b s ta n c e s, a n d c e ll d e b r is, ( x 2 7,5 0 0 ) these m aterials com prised a m ean 60 percent (40 to 76 percent) of arterial wall thickness. Age related changes in intramyocardial arterioles has been dem on strated by Billingham et al.1 The normal thickness of the subendothelial zones varied with age, ranging from 12.5 per cent in the 16 to 31 years age group, 29 percent in the 40 to 56 age years age group, and 37 percent in the 57 to 64 years age group. In patients with small vessel disease in our study, the thickness of the subendothelial zone was signifi cantly greater than the results of age related changes reported by Billingham et al1 and showed no correlation with age. M in im a l o r n o n - d i a g n o s t i c changes: T hese biopsy specim ens show ed no evidence of m yocellular hypertrophy or conspicuous interstitial fibrosis. Electron microscopic examina tion revealed only minimal myocardial changes. The pathologic classification of endo myocardial biopsy specimens from these 110 consecutive patients exam ined in our institution was tabulated in table I. There were 54 patients (49.1 percent) w ith pathological changes consistent with cardiomyopathy of whom 41 were idiopathic, five had doxorubicin cardiotoxicity, three had a history of alcohol abuse, two had h eart disease w hich occurred in the peripartal period, one had a family history of cardiomyopathy, and two had connective tissue disease, one with scleroderma and one with sys temic lupus erythematosus. Myocarditis was diagnosed in 12 patients (10.9 per cent). Amyloidosis was confirmed in two patients (1.8 percent). Ten patients (9.1

6 T A B L E I Incidence of Pathological Diagnosis in 110 Consecutive Patients Undergoing Endomyocardial Biopsy PATHOLOGY OF SMALL CORONARY VESSELS 185 Number o f P a tie n ts P ercent Cardiomyopathy Idiopathic 41 Adriamycin 5 Doxorubicin 3 Péripartum 2 Familial 1 Scleroderma 1 Lupus erythematosus 1 Myocarditis Amyloidosis Small vessel disease Hypertensive 5 Normotensive 11 Interstitial fibrosis Minimal or non-diagnostic changes percent) had only in terstitial fibrosis. Sixteen patients (14.6 percent) had significant intram ural small vessel abnormalities. Five of these patients had a history of hypertension but 11 patients had no known cause and w ere considered to be idiopathic. Sixteen patients (14.6 percent) showed minimal or non-diagnostic changes. II. Specimens consistent with the diagnosis of cardiomyopathy had the greatest degree of myocellular hypertrophy. The small vessel disease group had less myocellular hypertrophy b u t slightly m ore in te rstitia l fibrosis th an those w ith a pathologic diagnosis of cardiomyopathy. In the 16 patients with small vessel disease, there were no qualitative or quantitative differences in the morphological changes between patients with and without hypertension. T he m orphological changes in th e sm all vessels included m arked thickening of th e arterial wall owing to subendothelial deposits of heterogenous electron dense materials consisting of microfibrils, collagen and elastic fibers, cellular debris, and amorphous gran u lar substances. S u b endoth elial deposits com prised a m ean 60 p ercent (40 to 76 percent) of arterial wall thickness. Nine of these patients had additional m yocardial changes w hich w ere similar to those seen in cardiomyopathy. Four oth er patients showed interstitial fibrosis only and th e rem aining th ree patients had no conspicuous myocardial changes. M o r p h o m e t r ic A n a l y sis o f E n d o m y o c a r d ia l S p e c im e n s M orphom etric m easurem ents for each pathologic diagnosis are shown in table C l in ic a l Pa t h o l o g ic a l C o r r e l a t io n o f S m a l l V e s s e l D is e a s e The clinical presentation of patients w ith sm all vessel disease was varied. TABLE II Morphometric Measurements of Various Pathological Diagnosis on Endomyocardial Biopsy M y o c e llu la r H ypertrophy I n t e r s t i t i a l F ib r o s is D iam eter* S c o re s Area* S co res (fjm) P ercen t C ardiomyopathy Myocarditis Small vessel disease Hypertensive Normotensive Interstitial fibrosis Minimal of non-diagnostic changes ± ± ± ± ± ± *Mean ± standard deviation

7 186 PENG AND FRENCH T here w ere six m ales and 10 fem ales with a mean age of 53 years. Small vessel disease of th e m yocardium o ccu rred m ore fre q u e n tly in fem ale p a tie n ts, although overall more males underw ent endom yocardial biopsy. T here was no sig n ific a n t age d iffe re n c e b e tw e e n patients w ith small vessel disease and o th e rs u n d e rg o in g e n d o m y o c a rd ia l biopsy. Overall, eight patients presented with arrhythm ias, including two patients w ith associated h eart failure and one patient with associated chest pain. Five other patients presented with the recent onset of heart failure and three patients with chest pain. Thus, arrhythm ias were the most frequent clinical presentation in patients w ith sm all vessel disease. Hemodynamic studies revealed that 12 of these 16 patients with small vessel disease had a reduced left ventricular ejection fraction less than 50 percent. Discussion Although the most common indication for endomyocardial biopsy in this study was clinically suspected myocarditis, the actual incidence of biopsy proven myoc a rd itis was only 10.9 p e rc e n t. T he majority of our patients w ere diagnosed as having non-specific cardiom yopathy based on th e p resen ce of m yocellular hypertrophy, in terstitial fibrosis, and myofibrillar degeneration. The cause of cardiomyopathy in the majority of these patients was unknown. In other patients, cardiomyopathy was associated with certain clinical states, such as doxorubicin tr e a tm e n t, a lc o h o lism, p re g n a n c y, scleroderma, or systemic lupus erythematosis. Surprisingly, a significant num ber of patients were found to have small intramural coronary vessel abnormalities. In the p re se n t series of 110 consecutive patients, 16 patients (14.6 percent have been identified) with abnormal morphological changes in th eir small coronary vessels. This incidence of small vessel disease may be underestim ated because arterioles w ere not found in all biopsy specim ens. Som e 30 p e rc e n t of these patients with small vessel disease had a history of hypertension while 70 percent had no known associated conditions. The presence and significance of small vessel disease in coronary vessels is rather controversial. Relatively few reports relating the incidence and the clinical presentation of patients with small vessel disease of the myocardium are present in the literature. W eiss and Fenoglio17 reported 12 patients with histologic evidence of small vessel disease in a group of 100 p a tien ts. M cr eynolds and R o b erts13 described abnorm alities of intram ural c o ro n a ry a r te r ie s in 75 p e r c e n t o f patients with hypertrophic cardiomyopathy. Richardson et al14 dem onstrated the presence of abnorm al intram ural small arteries and arterioles in patients who had undergone endomyocardial biopsy. Some of the controversy concerning the incidence of small vessel disease may be related to evaluation of light microscopic specim ens only, since th e d eg ree of in v o lv em en t in sm all vessels m ay be appreciated b etter by electron m icroscopic examination. Small arterial changes are frequently seen in th e k id n ey in p a tie n ts w ith hypertension, but the role of hypertension in th e p ro d u ctio n of in tram u ral small coronary vessel changes is controversial. Donomae et al4 have reported in p atients w ith hyperten sio n th at intramyocardial arteries w ere rarely involved with the type of lesions that w ere seen in small vessels of the kidney. In contrast, Blumenthal et al2 described a hem odynamic lesion in small coronary arteries in p a tie n ts w ith h y p e rte n sio n. This lesion consisted of a fibrous or fibroblastic intimai thickening with a variable PAS reactio n and in arterio les as hyaline th ick en in g w ith PAS positive fibrils. T h ese h em o d y n am ic le sio n s w ere

8 PATHOLOGY OF SMALL CORONARY VESSELS 187 thought to be associated with hypertension. In our p resen t study, five of 16 patients with small vessel disease had a history of hypertension, suggesting that a hemodynamic factor may contribute to the pathogenesis of small coronary vessel changes. On the other hand, 11 of 16 patients had no significant associated conditions and, thus, the etiology of this id io p a th ic sm all v e sse l d ise a se was unknown. O ther etiologies have been suggested as a cause of small vessel disease. There may be a causative relationship betw een an episode of m yocarditis and subsequent development of small vessel disease. Coxsackie B viral infection has been shown to produce extensive arteritis and capillary damage in experim ental anim als.3,15 Jam es9 also has suggested that small coronary vessel disease may be causative for some obscure cardiomyopathies. Ham by et al8 found lesions similar to those described by Blum enthal et al2 but in patients with cardiomyopathy who w ere not hypertensive. Small coronary arteriolar changes may represent arteriolar cardiosclerosis, part of a generalized process of arteriosclerosis. Factor and Sonnenblick6 have suggested that spasm of small coronary vessels may lead to myocardial damage and cardiomyopathy. Secondary sm all vessel disease has been described in association with diabetes mellitus, amyloidosis, and connective tissue diseases.7,17 Small vessel disease was shown to be 2.5 tim es more frequent in diabetics than in non diabetic p atients.2 The basem ent m em brane of small vessels in diabetic patients was freq u en tly found to be th ick en ed up to 1000 angstrom s.17 None of our patients with small vessel disease had a history of diabetes mellitus. Moreover, no significant thickening of basem ent m em branes of small coronary vessels was observed in th e se p a tie n ts. Two p a tie n ts in our present series with amyloidosis had amyloid fibrils predom inently accum ulated aro u n d v essels an d along b a se m e n t m em branes. Small vessel disease may also be associated with an immunologically mediated process, such as connective tissue disease; however, none of our cases employing direct im m unofluorescence had any evidence of immune complex deposition along the vessel walls or elsewhere. The pathogenetic mechanisms of small vessel disease have not been totally elucidated. Factor5 has dem onstrated that the prim ary damage was endothelial. It is conceivable that hem odynam ic insult, alcohol, chemical toxin, viral infection, or im m unological m ediated processes m ay d a m a g e e n d o th e liu m c a u sin g increased perm eability and induce vascular sm ooth m uscle cells to produce collagen, elastin, and basem ent membrane-like substance, resulting in sclerotic lesions similar to those shown in our present study. Patients with small vessel disease may present with a variety of clinical presentations including arrhythm ias, congestive heart failure, chest pain, and sudden death. Arrhythmias w ere the most frequent presentation in our patients with small vessel disease. Jam es10 has proposed that lesions in small vessels supplying the critical pacem aking or cond u c t i n g t i s s u e c o u ld r e s u l t in arrhythm ias. T he association of chest pain, normal epicardial coronary arteries, and small vessel disease has been investigated by several g ro u p s.11,12,14 Koch e t al11 have d e m o n stra te d th a t small vessel disease in endomyocardial biopsy specimens in a group of patients w ith chest pain and norm al coronary arteries. Vernauskas et al16 have studied p a tie n ts w ith card io m y o p ath y using angiocardiography and found diffuse involvement of small coronary arteries. The pathological diagnosis of cardiomyopathy is based on several non-specific morphological changes. Therefore,

9 188 PENG AND FRENCH cardiomyopathy may represent a heterogenous group of diseases with similar myocardial abnormalities. Based on this study, small vessel disease may be one of many causes of cardiomyopathic process, since m ore th an h alf of th ese biopsy specimens showing small vessel abnormalities w ere associated with myocardial changes similar to those seen in cardiomyopathy, while others had interstitial fibrosis or minimal myocardial changes. These m yocardial changes may rep resent various stages of involvem ent from sm all v essel d isease. T his g ro u p of patients with small vessel disease can be separated from those with cardiomyopathy of unknown cause. It has been suggested that the association of small vessel disease and cardiomyopathy may carry a poor prognosis, however, further studies are necessary to substantiate this observation. Conclusion (1.) The incidence of small coronary v e sse l d is e a s e in 110 c o n s e c u tiv e p a tien ts u n d e rg o in g endom yocardial biopsy was 14.6 percent. (2.) A rrhythmias were the most frequent clinical presentation follow ed by recen t onset of heart failure and chest pain. (3.) TJltrastructurally, the subendothelial zone was m arkedly thickened and com posed of heterogenous electron dense materials w hich may result in loss of vessel wall compliance and luminal narrowing. (4.) The consequence of these changes may lead to myocardial damage. References 1. B i l l in g h a m, M. E., S c h w a r t z, B., R u d e r, A., and H a r r i s o n, D.: A stu dy of ag e-re lated changes in intram yocardial arterioles. Lab. Invest. 36:331, B l u m e n t h a l, H. T., A l e x, M., and G o l d e n g e r, S.: A study of the intramural coronary artery branches in diabetes mellitus. Arch. Pathol. 70:13-28, B u r c h, G. E., T s n i, C. Y., and H a r r, J. M.: Pathological changes of aorta and coronary arteries of mice infected with Coxsackie B4 virus. Proc. Exp. Biol. Med. 137: , D o n o m a e, I., M a t s u m o t o, Y., and U e d a, E.: Significance of coronary arteriosclerosis in the intram uscular coronary arteries. G eriatrics 20: , F a ctor, S. M.: Intramyocardial small vessel disease in ch ro n ic alcoholism. Am. H e a rt J. 92: , F a c t o r, S. M. an d S o n n e n h l i c k, E. H.: H y pothesis: Is congestive cardiom yopathy caused by a hyperreactive myocardial microcirculation (microvascular spasm)? Am. J. Cardiol. 50: , G e e r, J. C., B i s h o p, S. P., and J a m e s, T. N.: Pathology of small intramural coronary arteries. Pathol. Ann. 24: , H a m b y, R. I., Z o n e r a i c h, S., and S h e r m a n, L.: Diabetic cardiomyopathy. J. Am. Med. Assoc. 229: , J a m e s, T. N.: An etiologic concept concerning the obscure myocardiopathies. Prog. Cardiovasc. Dis. 7:43-64, J a m e s, T. N.: The role of small vessel disease in myocardial infarction. Circulation 39,40 (suppl. IV): 13-19, K o c h, F., B i l l i n g h a m, M., R i d e r, A., M a s o n, J. W., C i p r i a n o, P. R., a n d H a n c o c k, E. W.: P a th o p h y s i o l o g y o f a n g i n a w i t h n o r m a l c o r o n a r y a n g i o g r a m s. C i r c u l a t i o n 54 ( s u p p l. 2):173, M a s o n, J. W. and S t r e f l i n g, A.: Small vessel disease in the heart resulting in m yocardial necrosis and death despite angiographically normal coronary arteries. Am. J. Cardiol. 44: , M c R e y n o l d s, R. A., and R o b e r t s, W. C.: The intramural coronary arteries in hypertrophic cardiomyopathy. Am. J. Cardiol. 3 5 :1 5 4, R i c h a r d s o n, P. J., L i v e s l e y, B., O r a m, S., O l s e n, E. G. J., and A r m s t r o n g, P: Angina pectoris with normal coronary arteries. Transvenous myocardial biopsy in diagnosis. Lancet 2: , S o h a l, R. S., B u r c h, E. E., C h u, K. C., L e i - d e r m a n, E., and COLCOLOUG, H. L.: Ultrastructural changes in cardiac capillaries of Coxsackie virus B 4 infected mice. Lab. Invest. 19: , V a r n a u s k a s, E., I v e m a r k, B., P a u l i n, S., and R y d e n, B.: Obscure cardiomyopathies with coronary artery changes. Am. J. Cardiol. 29: , W e i s s, M. B. and F e n o g l i o, J. J.: Small vessel disease: Fact or fiction. Endomyocardial Biopsy: Techniques and Applications. Fenoglio, J. J., ed. B oca R ato n, F L, CRC P re ss, 1983, pp