3 Clin Pathol 1993;46:32-36

Collagen remodelling in myocardia of patientswith diabetes

M Shimizu, K Umeda, N Sugihara, H Yoshio, H Ino, R Takeda,Y Okada, I Nakanishi

AbstractAims: To investigate collagen remodellingin the interstitium of the heart in patientswith diabetes.Methods: Immunohistochemical study ofthe biopsied myocardium using type spe-cific anticollagen antibodies (I, III, IV, V,VI) was performed in 12 patients withnon-insulin dependent diabetes mellitusand six non-diabetic patients. There wasno history of hypertension or coronaryartery stenosis in any of the patients.Results: Noticeable accumulations of col-lagen types I, III, andVI in the myocardialinterstitium were recognised in bothgroups, but little accumulation oftypes IVor V was found. Types I and III mainlystained in the perimysium and perivas-cular region, while type VI predominantlystained in the endomysium. There was nodisease specific accumulation of collagenin diabetes mellitus. The percentage oftotal interstitial fibrosis in the myocar-dium was significantly higher in the dia-betic group than in the control group(p < 0.05). Although the percentages ofcoliagen types I and VI did not differbetween the two groups, the percentagetype of III was significantly higher in thediabetic group than in the controls(p < 0-01).Conclusions: Collagen remodelling mainlyas a result of an increase in collagen typeIII in the perimysium and perivascularregion, occurs in the hearts of patientswith diabetes.

(X Clin Pathol 1993;46:32-36)

Second Department ofInternal MedicineM ShimizuK UmedaN SugiharaH YoshioH InoR TakedaFirst Department ofPathology, School ofMedicine, KanazawaUniversity, JapanY OkadaI NakanishiCorrespondence to:Dr Masami Shimizu, TheSecond Department ofInternal Medicine, School ofMedicine, KanazawaUniversity, Takara-machi13-1, Kanazawa 920, JapanAccepted for publication7 August 1992

Primary myocardial injury has been shown indiabetes mellitus even in the absence of hyper-tension and coronary atherosclerosis.' 2 Thehistopathological changes found in the myo-

cardium of patients with diabetes includemyocyte hypertrophy, interstitial fibrosis, andperivascular fibrosis. Interstitial fibrosis iscomposed largely of fibrillar collagen. Regan etal reported the presence of interstitial collagendeposition in left ventricular biopsy specimensin diabetic patients, and found collagen accu-mulation in perivascular loci, between myo-fibres, or as replacement fibrosis in a

postmortem study of cardiac tissue of dia-betics.3 On the other hand, it has beenreported that many phenotypes with differingmolecular structures are present in the fibrillarcollagen, with the distribution of these pheno-

types varying in different tissues.4 5Weber et alcarried out several studies on myocardial colla-gen remodelling.6`8 However, no informationis available on the changes in collagen in themyocardium of patients with diabetes. Accord-ingly, we investigated the localisation of colla-gen phenotypes in the interstitium of suchcardiac tissue and estimated them quantita-tively using an immunohistochemicalmethod.

MethodsBiopsy specimens were obtained from 12patients with non-insulin dependent diabetesmellitus who had undergone cardiac catheter-isation because of complaints of chest pain orelectrocardiographic abnormalities. In allpatients the absence of more than 25% coro-nary artery stenosis was documented angio-graphically, and there was no history ofhypertension, chronic renal failure, or otherendocrinological disorders. Biopsy specimensfrom six non-diabetic patients, in whom car-diac catheterisation including endomyocardialbiopsy was performed as. part of the evaluationof arrhythmia or chest symptoms, but in whomno clinically relevant coronary artery stenosisor abnormal haemodynamics was found,served as controls. Diabetes mellitus was diag-nosed and classified in accordance with thecriteria of the National Diabetes Data Group.9The diabetic patients comprised six men andsix women (mean (range) age 52-1 (2-9)years). The controls comprised three men andthree women with a mean age of 43-5 (3.7)years. The duration of diabetes mellitus rangedfrom one to 17 years (mean 10-5 years). Thedegree of diabetic complications ranged frommild to moderate. A right ventricular endo-myocardial biopsy was performed using theKonno-Sakakibara bioptome or Bipal biopsyforceps (Cordis), and specimens were obtainedfrom the region of the interventricular septum.Informed consent was obtained from eachpatient before cardiac catheterisation.

HISTOPATHOLOGICAL STUDY

The specimens obtained by endomyocardialbiopsy were submerged immediately in 10%buffered formalin. After being embedded inparaffin wax they were cut into 4 pm sectionson a longitudinal plane and more than eightcontinuous sections were obtained from eachspecimen. One of these sections was stainedwith haematoxylin and eosin, another wasstained with Mallory-Azan; the other sectionswere stained with type specific anti-collagen

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Myocardial collagen in diabetics

antibodies or normal rat IgG as a negativecontrol.

IMMUNOHISTOCHEMICAL STUDYSections of paraffin wax embedded myocar-dium were treated with 0-05%,protease typeXXIV (Sigma, USA) for 30 minutes; non-specific reactions were checked for with 0-3%hydrogen peroxide. The prepared sections werestained by the avidin-biotin-complex (ABC)method'0 using type specific anticollagen anti-bodies (types I, III, IV, V, VI) which had beenpurified. "

LOCALISATION OF COLLAGEN TYPESMyocardial fibrosis in the sections stained withMallory-Azan was classified into three compo-nents: (1) endomysial fibrosis-fibrosis provid-ing lateral cell to cell connections; (2)perimysial fibrosis-fibrosis surrounding myo-cytes to form muscle bundles; (3) perivascularfibrosis-fibrosis surrounding intramyocardialsmall vessels 10,um-50 pm in diameter. Weused the stain of the endocardium to identifythe potential effects on staining quality causedby fixation of the specimen, density of theantibodies, and other technical problems. Thedistribution of each type of collagen in eachregion was assessed by three cardiac patholo-gists, based on the following system: (-) nostaining; (1 + ) only slight staining; (2 + ) some-what intense staining, but less intense than thatof the endocardium; (3 +) intense stainingequivalent to that of the endocardium. Bloodvessels 10-50 ,um were found in biopsy speci-mens obtained from nine (two controls andseven diabetic patients) out of the 18 subjects,with perivascular fibrosis evaluated in thesespecimens.

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QUANTIFICATION OF TOTAL MYOCARDIALINTERSTITIAL FIBROSIS AND VARIOUS TYPES OFCOLLAGENThe extent of total fibrosis and various types ofcollagen was measured by the point countingmethod" 13using the sections stained withMallory-Azan and each type specific anti-collagen antibody. We used a point countingmethod grid with the points arranged in asquare pattern and a distance of 10 ,umbetween points. More than 2000 points, whichlay on the myocardial cells or on the fibrotictissue stained blue by Mallory-Azan and brownby type specific anticollagen antibodies only inthe longitudinally cut cells, were recognised.The percentage of fibrosis was calculatedaccording to the formula:

percentage of fibrosis =

points lying on the fibrosis

points lying on the myocytes and fibrosisx 100.

All data were expressed as mean (1 SEM).Statistical analysis was performed using Stu-dent's unpaired t test and the Mann-WhitneyU test. Differences were considered statis-tically significant when the p value was lessth3u 0-05.

ResultsSubstantial accumulations of collagen types I,III, VI in the myocardial interstitium wererecognised in both groups (figs 1-3). On theother hand, no accumulation of collagen typesIV orV was found in either group, except for aslight accumulation of collagen type IV in theintima of the small vessels. Collagen type I wasmainly stained in the perimysium and perivas-

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Figure I Perimysial fibrosis stained with Mallory-Azan (upper left) and immunostained with antibodies to type I(upper right), type III (lower left) and type VI (lower right) collagens.

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Shimizu, Umeda, Sugihara, Yoshio, Ino, Takeda, Okada, Nakanishi

Figure 2 Endomysialfibrosis stained withMallory-Azan (upper left)and immunostained withantibodies to type I (upperright), type III (lower left)and type VI (lower right)collagens.

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cular region, but only slightly stained in theendomysium. Collagen type III was intenselystained in the perimysium and perivascularregion and weakly stained in the endomysium.Collagen type VI was intensely stained in theendomysium and weakly stained in the perimy-sium and perivascular region (fig 4). There wasno disease specific accumulation of collagen indiabetes mellitus.The percentage of fibrosis in the sections

stained with Mallory-Azan was significantlyhigher in the diabetic group than in the controlgroup (17-0 (2 8) v 7-2 (1-9)%; p < 0 05).Figure 5 shows a comparison of the percentageof collagen types I, III, VI in the two groups.The percentages of collagen types I and VI did

not differ significantly between the two groups(type I:diabetes mellitus 11-4 (3-0); controls4-3 (1-3)%; type VI:diabetes mellitus 24-5(2-5); controls 22-0 (3 4)%). On the otherhand, the percentage of collagen type III indiabetes mellitus was significantly higher thanthat of the controls (19-6 (2 3) v 7-8 (3 3)%;p < 0.01).

DiscussionInterstitial fibrosis was significantly increasedin the group with diabetes mellitus. Most ofthis was composed of collagen fibres. Severaltypes of differing molecular structures arepresent in these collagen fibres, but it is not

Figure 3 Perivascularfibrosis stained withMallory-Azan (upper left)and immunostained withantibodies to type I (upperright), type III (lower left)and type VI (lower right)collagen.

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Myocardial collagen in diabetics

Type III

M 3--) (1-)

(2 -)

Figure 4 Semiquantitative analysis of the distribution of collagen typePM = perimysium, EM = endomysium, PV = perivascular region.(-) not detectable, (I+) trace, (2+) moderate, (3+) abundant.

Figure 5 Comparison ofthe percentages of collagentypes I, III and VI in thetwo groups. The percentageof collagen type III indiabetes mellitus was

significandy higher thanthat of the controls.

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clear whether differences in thproportion of these collagen t3hearts of patients with diabete

Differences were found in theach type of collagen among tthis distribution was similar iCollagen types I and III are r

interstitial fibrosis. Borg et all4et al l' reported results sirobtained in the present study,the localisation of collagen tyAthe diabetic heart is not sj

disease.No differences in the amoui

Type VI VI were found between the diabetic andm** r** ~ control groups, but type III was significantly

increased in patients with diabetes. The mech-anism involved is unknown, but myocardialischaemia is presumed to have a role. In thehearts of patients with diabetes luminal nar-rowing due to intramural coronary arteriolewall thickening or deposition of periodic acidSchiff positive material and perivascular fibro-sis are present. As a result the diabetic myocar-dium is exposed to ischaemia. As Medugorac et_al' speculated about hypertrophied cardiactissue, the diminished blood flow in the dia-betic myocardium over extended periods may

PM EM PV lead to myocyte necrosis and subsequent heal-ing, resulting in increased synthesis of type IIIcollagen by cardiac fibroblasts. On the otherhand, Villee et al,'7 in experiments usinghuman skin fibroblasts cultured in vitro, foundan increased glucose concentration in the

7s I, III and VI. medium and increased production of collagenStaining pattern: type III. This interesting report suggests that in

diabetes mellitus, in addition to the develop-ment of ischaemia due to microangiopathy, theincreased glucose concentration may itselfinfluence collagen metabolism. Due to theincrease in blood glucose, fibrillar collagenbegins to accumulate in the adventitia of

lintramural coronary arteries and then extendsinto the intramuscular spaces; this is followedby an abnormality in the permeability of theseintramural vessels.'8 An increase in coronaryvascular permeability may also facilitate theinflux of growth factors in the interstitium andlead to further fibroblast proliferation andenhanced collagen synthesis. Type III collagenin myocardial interstitium may then beincreased by the above mentioned mecha-nisms. In addition to cardiac fibroblast pro-liferation or enhanced collagen synthesis,collagen degradation may be retarded,although no information is available about this.Other interesting questions include which fac-tors determine collagen synthesis in the cardiactissues of patients with diabetes (for example,in addition to angiotensin II and aldosteronereported in the pressure overloaded left ven-tricle,"8 '9 the involvement of glucose, insulin,etc.) and what kind of drug might inhibit thisprocess. It is expected that ongoing studiesusing molecular biological techniques will shedlight on these issues.

Collagen is the most important of theDiabetic extracellular structural proteins, with collagenmyocardium

fibres acting as lateral connections betweencells and muscle bundles to govern archi-

Le localisation or tecture, maintain shape and wall thickness,ypes exist in the and resist myocardial deformation. However,s. while collagen is the major determinant ofe distribution of myocardial stiffness,20 21 collagen phenotype:issues, although remodelling may also influence cardiac func-in both groups. tion. Bouchard et al22 in an echocardiographicepresentative of study, reported a reduced left ventricular end-and Shekhonin diastolic volume index and impaired left en-milar to those tricular diastolic filling in diabetics,23 and,suggesting that Regan et al' also reporting increased leftpes I and III in ventricular chamber stiffness. We surmise thatpecific for this the increase in interstitial fibrosis and altera-

tions in collagen phenotypes found in thents of types I or present study may in some way be related to

Type

ox0

0 PM EM PV

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these changes in cardiac function, althoughfurther detailed investigations will be neededto clarify this.

1 Rubler S, Dlugash J, Yuceoglu YZ, Kumral T, BranwoodAW, Grishman A. New type of cardiomyopathy associatedwith diabetic glomerulosclerosis. Am Cardiol 1972

30:595-602.2 LedetT Diabetic cardiomyopathy. Quantitative histological

studies of the heart from young juvenile diabetics. ActaPathol Microbiol Scand (Sect A) 1976;84:421-8.

3 Regan TJ, Lyons MM, Ahmed SS, et al. Evidence forcardiomyopathy in familial diabetes mellitus. Clin Invest1977;60:885-99.

4 Miller EJ. The structure of fibril-forming collagens. Ann NYAcad Sci 1985;460:1 -13.

5 Kuhn K. The collagens-molecular and macromolecularstructures. In: Tschesche H, ed. Proteiznases in i.nflanima-tinon anid tumslouir invasion. Berlin: Walter de Gruvter & Co.,1986:107-43.

6 Weber KT, Janicki JS, Shroff SG, Pick R, Chen RM, BasheyRI. Collagen remodelling of the pressure-overloaded,hypertrophied nonhuman primate myocardium. Circ Res1988;62:757-65.

7 Jalil JE, Doering CW, Janicki JS, Pick R, Shroff SG, WeberKT. Fibrillar collagen and myocardial stiffness in theintact hypertrophied rat left ventricle. Circ Res 1989;64:1041 -50.

8 Weber KT, Pick R, Silver MA, et al. Fibrillar collagen andremodelling of dilated canine left ventricle. Circulation1990;82:1387-401.

9 National Diabetes Data Group. Classification and diagnosisof diabetes mellitus and other categories of glucoseintolerance. Diabetes 1979;28:1039-57.

10 Hsu S, Raine L, Fanger H. A comparative study of theperoxidase-anti-peroxidase method and an avidin-biotincomplex method for studying polypeptide hormones withradioimmunoassay antibodies. Ani .7 Clin Pathol 1981;75:734-8.

11 MinamotoT, OoiA, OkadaY, Mai M, NagaiY, Nakanishi I.Desmoplastic reaction of gastric carcinoma: A light- andelectron-microscopic immunohistochemical analysisusing collagen type specific antibodies. Huni Pathol1988;19:815-21.

12 Sugihara N, Genda A, Shimizu M, et al. Quantification ofmyocardial fibrosis and its relation to function in essentialhypertension and hypertrophic cardiomyopathy. Cliii Car-diol 1988;11:771-8.

13 Kita Y, Shimizu M, Sugihara N, et al. Correlation betweenhistopathological changes and mechanical dysfunction indiabetic rat hearts. Diabetes Res Clin Pract 1991; 11:177-88.

14 BorgTK, Gay RE, Johnson LD. Changes in the distributionof fibronectin and collagen during development of theneonatal rat heart. Collagen and Related Research1982;2:211-8.

15 Shekhonin BV, Domogatsky SP, Idelson GL, KotelianskyVE. Participance of fibronectin and various collagen typesin the formation of fibrous extracellular matrix in cardio-sclerosis. JMol Cell Cardiol 1988,20:501-8.

16 Medugorac I, Jacob R. Characterisation of left ventricularcollagen in the rat. Cardiovasc Res 1983;17:15-21.

17 Villee DB, Powers ML. Effect of glucose and insulin oncollagen secretion by human skin fibroblasts in vitro.Nature 1977;268:156-8.

18 Weber KT, Brilla CG. Pathological hypertrophy and cardiacinterstitium. Fibrosis and renin-angiotensin-aldosteronesystem. Circulation 1991 ;83: 1849-65.

19 Brilla CG, Pick R, Tan LB, Janicki JS, Weber KT.Remodelling of the rat right and left ventricles inexperimental hypertension. Circ Res 1990;67: 1355-64.

20 BorgTK, RansonWF, Moslehy FA, Caulfield JB. Structuralbasis of ventricular stiffness. Lab Invest 1981 ;44:49-54.

21 BorgTK, Caulfield JB. The collagen matrix of the heart. FedProc 1981;40:2037-41.

22 Bouchard A, Sanz N, Botvinick EH, et al. Non-invasiveassessment of cardiomyopathy in normotensive diabeticpatients between 20 and 50 years old. Ani 7 Med1989;87:160-6.

23 Shimizu M, Sugihara N, KitaY, Shimizu K, Shibayama S,Takeda R. Increase in left ventricular chamber stiffness inpatients with non-insulin dependent diabetes mellitus.Jpn Circ _ 1991;55:657-64.

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