One of the main features in human tetralogy of Fallot (TF) is right ventricular hypertrophy (RVH) due to pressure (sub-pulmonary stenosis) and volume overload (ventricular septal defect). Currently, primary correction at a young age is the treatment of choice. To unravel the role of extracellular matrix in RVH, we examined myocardial expression of collagens and fibronectin in TF patients with primary correction (TF1, age 0.7 ± 0.2 yr,), secondary surgery (TF2, age 36.9 ± 4.6 yr), and in age-matched control patients. Sirius red staining quantified by video imaging showed significantly increased interstitial staining for collagens in both TF1 and TF2 groups as compared to respective controls. Fibronectin was expressed in extracellular spaces, perivascular regions, and in some cardiomyocytes. Quantitative analysis of fibronectin revealed increased expression in only TF1 group as compared to respective control. Our results indicate an increased amount of myocardial extracellular matrix deposition as a sign of fibrosis during RVH in patients with TF

Bogers, A.J.J.C. (Ad)

Peters, T.H.F. (Erik)

Sharma, H.S. (Hari)

Yilmaz, P. (Pinar)

Erasmus University Digital Repository

278Quantitative Analysis of Collagens and Fibronectin Expression in Human Right Ventricular HypertrophyaT.H.F. PETERS,b,c H.S. SHARMA,b,d E. YILMAZ,b AND A.J.J.C. BOGERScbPharmacology, cCardiothoracic Surgery, Erasmus University Medical Center, Rotterdam, The Netherlands ABSTRACT: One of the main features in human tetralogy of Fallot (TF) isright ventricular hypertrophy (RVH) due to pressure (sub-pulmonary stenos-is) and volume overload (ventricular septal defect). Currently, primary correc-tion at a young age is the treatment of choice. To unravel the role ofextracellular matrix in RVH, we examined myocardial expression of collagensand fibronectin in TF patients with primary correction (TF1, age 0.7  0.2 yr.),secondary surgery (TF2, age 36.9  4.6 yr), and in age-matched control pa-tients. Sirius red staining quantified by video imaging showed significantly in-creased interstitial staining for collagens in both TF1 and TF2 groups ascompared to respective controls. Fibronectin was expressed in extracellularspaces, perivascular regions, and in some cardiomyocytes. Quantitative analy-sis of fibronectin revealed increased expression in only TF1 group as comparedto respective control. Our results indicate an increased amount of myocardialextracellular matrix deposition as a sign of fibrosis during RVH in patientswith TF.INTRODUCTIONTetralogy of Fallot (TF) is a common form of cyanotic congenital heart disease,with an incidence rate of 1 out of 2,000 newborns. TF is characterized by overridingof the aorta, a ventricular septal defect, (sub)valvular pulmonary stenosis, and rightventricular hypertrophy (RVH), resulting in hypoxemia due to diminished pulmo-nary flow.1 The RVH in human TF is an adaptive response of cardiomyocytes to in-creased pressure as well as volume overload caused by (sub)pulmonary stenosis andby ventricular septal defect, respectively. The adaptive response requires proportion-ate increase of coronary flow or growth of the coronary vasculature, leading to my-ocardial remodeling as well as development of fibrosis and, without treatment,ultimately results in cardiac failure.2−5In myocardial fibrosis, collagens and fibronectin play a prominent role. Damageof the collagen structure compromises the myocyte support, decreases myocardialstrength and stiffness, and allows expansion of the tissue.6 Collagen fibers are orga-nized in the collagen network, which is found in the extracellular space of the myo-aThis work is supported by the Netherlands Heart Foundation (NHS 96.082).dAddress correspondence to: Hari S. Sharma, Ph.D., Institute of Pharmacology, Erasmus Uni-versity, P.O. Box 1738, 3000 DR Rotterdam, The Netherlands; Telephone: 31 10 4087963; Fax:31 10 4089458; E-mail: SHARMA@FARMA.FGG.EUR.NL279PETERS et al.: COLLAGEN AND FIBRONECTIN EXPRESSIONcardium.7−10 In myocardial fibrosis a disproportionate accumulation, either reactiveor reparative, of collagen has been observed. Experimental11 and clinical data12,13show that a rise in collagen content increases myocardial stiffness and promotes ab-normalities of cardiac function during hypertrophy. In a morphometrical study, anincrease in fibrosis in TF was associated with an increased diameter of myocytes andan increase of myocardial disarray.14 In addition, the perivascular accumulation ofcollagen fibers may impair the vasodilator capacity of intramyocardial coronary ar-teries and may contribute to the decrease in coronary reserve.15Fibronectin is found in the extracellular matrix of most tissue, serving as a bridgebetween cells and interstitial collagen mesh network and influencing diverse pro-cesses, including cell growth, adhesion, migration, and wound repair.16 In rats, fetalfibronectin was shown to have accumulated during development of cardiac hypertro-phy by pressure overload.17 The hypertrophied heart exhibits significant qualitativeas well as quantitative changes in gene expression. The altered expression patternsin hypertrophy include transient expression of proto-oncogenes.18 It is thought thatthose changes in cardiac hypertrophy reflect a change toward an embryonic programof gene expression.19 However, it is still not clear whether those altered expressionpatterns are general markers of cardiac hypertrophy or markers of a specific parallelpathogenic process, such as TF. Animal studies have shown that the levels of mRNAfor fibronectin and collagens were elevated in cardiac hypertrophy.17Most attention in research on human TF has so far focused on clinical treatmentand surgical methods to limit the myocardial injury. TF patients are usually operatedon during their first year of life. The aim of early primary repair is to close the ven-tricular septal defect and to remove the right ventricular outflow tract obstruction,thus relieving the hypoxemia and eliminating the stimulus for the adaptive RVH aswell as preserving right ventricular function.1,20 However, after corrective surgerysome patients develop right ventricular failure due to volume-overload caused bypulmonary regurgitation and may for this reason require further secondary correctivesurgery later in life.21,22 Histopathological studies on TF myocardial tissue reportedthat the myocardial cell diameter was reduced in patients after corrective surgery, in-dicating regression of RVH. Nevertheless, it is not yet known to what extent the RVHin TF patients regresses. Little attention has been paid to the molecular componentof this disease. In this study, we examined the degree of fibrosis in human TF bymeasuring the expression of myocardial fibrosis markers, such as fibronectin andcollagen, in right ventricular biopsies obtained during corrective surgery for TF andcomparing the data with that of age-matched patients with normal right ventricle.MATERIALS AND METHODSBiopsiesThe present study was approved by the Medical Ethical Committee of the Uni-versity Hospital, Erasmus University, Rotterdam. Myocardial tissue biopsies wereobtained from patients who underwent primary corrective surgery (TF1, mean age0.7 ± 0.2 yr, N = 11), secondary surgery (TF2, mean age 36.9 ± 4.6 yr, N = 6), andage-matched control patients. All control patients had clinically normal right ventri-cle and underwent autograft aortic root replacement (C1, mean age 1.5 ± 0.2 yr, N =280 ANNALS NEW YORK ACADEMY OF SCIENCES6 and C2, mean age 31.7 ± 4.2 yr, N = 12). Myocardial tissue was fixed in 4%paraformaldehyde in PBS for minimal 24 h and further processed for dehydrationand embedding in paraffin for histological and immunohistochemical studies.23 Sirius Red StainingThe total collagen in myocardial tissue specimens was stained with Sirius redF3BA.24 Tissue sections of 6 µm thickness, were treated with 0.2% aqueous phos-phomolybdic acid and incubated in 0.1% Sirius red. Before dehydration, the slideswere treated with 0.01N HCl and mounted. The Sirius red–stained area was deter-mined as percentage of total tissue area using a Kontron-KS 400 (KontronElectronik/Zeiss, Eching, Germany) computerized image analysis system. Distribu-tion of collagen fibers in the interstitial space as well as in the perivascular regionwas measured as Sirius red–positive area. Twelve different images from each sectionwere analyzed to calculate the mean percentage of stained tissue area and statistical-ly significant values were accepted at p ≤ 0.05.ImmunohistochemistryAfter fixation and dehydration, the cardiac tissue was embedded in paraffin. Sec-tions of 6 µm thickness were cut and mounted on poly-L-lysine–coated microscopeslides, followed by immunohistochemistry using the avidin-biotin complex (ABC)method (BioGenex, San Ramon, CA). The sections were deparaffinized andquenched for endogenous peroxidase by 2% hydrogen peroxide in methanol. Non-specific binding sites were blocked by 10% normal goat serum. Human serum pre-absorbed polyclonal antibodies, against human fibronectin in 1:1,000 dilution (LifeTechnologies, Breda, The Netherlands), were applied as primary antiserum and thesections were incubated at room temperature for 30 min. Negative controls were per-formed by omission of the primary antibody. After washing in 0.5% Tween-20 inPBS solution, the sections were incubated with mouse biotinylated anti-rabbit IgG(BioGenex, San Ramon, CA) for 30 min and subsequently, with peroxidase conju-gated streptavidin. Color was developed using 3,3′-diaminobenzidine tetrahydro-chloride dehydrate (DAB) and then sections were counter-stained with hematoxylin.Sections were mounted and visualized under the light microscope and photographed.A semiquantitative analysis of the sections was done by two independent observersusing a staining score ranging from 0 (no staining) to 4 (very strong, dark brown,staining) to assess the level of fibronectin expression in the myocardial tissue ob-tained from TF patients and age-matched controls.FIGURE 1. Right ventricular total collagen content in patients with tetralogy of Fallot.Micrographs of right ventricular tissue obtained from patients undergoing surgery (A) forautograft aortic root replacement (Control), (B) for primary repair due to tetralogy of Fallot(TF1), and (C) for secondary surgery (TF2). Tissue sections were prepared and stained withcollagen-specific stain, Sirius red. Collagen fibers appear red and are localized in interstitialand perivascular areas. Note the interstitial fibrosis in the case of TF patients. Bar = 250 µm.(D) Significantly enhanced interstitial collagen content in TF1 group as compared to thecontrol. Distribution of collagen fibers in tissue was visualized under normal and polarizedlight and quantified using video imaging software. Values are fold induction and shown asmean ± SEM of sirius red–positive interstitial/total tissue area (arrows).281PETERS et al.: COLLAGEN AND FIBRONECTIN EXPRESSION282 ANNALS NEW YORK ACADEMY OF SCIENCESRESULTS AND DISCUSSIONSirius red staining was performed for the total collagens in order to assess the de-gree of myocardial fibrosis. The red staining was quantified by video imaging andshowed a significantly increased interstitial and perivascular staining for total col-lagens in TF1 (1.4 ± 0.2 fold) (FIG. 1A and D) and TF2 (1.6 ± 0.2 fold) (FIG. 1B and1D) as compared to their respective controls (FIG. 1C and 1D). The immunolocal-ization of fibronectin showed that fibronectin was expressed in the interstitium, inthe perivascular area, as well as in some cardiomyocytes. Semiquantitative analysisusing a staining score ranging from 0−4, revealed that the fibronectin expression wassignificantly increased in TF1 (FIG. 2A and 2D) as compared to control (3.0 ± 0.2versus 2.6 ± 0.2). There were no differences in fibronectin expression in patientsfrom TF2 (FIG. 2B and 2D) as compared with age-matched controls (FIG. 2C and2D). Our results indicate an increased amount of myocardial extracellular matrixdeposition in RVH in patients with TF, with an apparent increase of total collagenand a slight increase in fibronectin at young age. The apparent development of cardiac hypertrophy is induced by changes in car-diac gene expression that provide the heart a means of compensation for increasedhemodynamic load.25 In this regard, our data are in agreement with the histopatho-logic findings from the RV myocardium in preoperative TF, which show interstitialfibrosis, myofibrillar disorganization, disarray, and degenerative changes, in addi-tion to myocardial cell hypertrophy.18,26,27 The changes found in our study of ex-pression of collagens and fibronectin could be attributed to altered structure orfunction of the myocardium. It is believed that changes in gene expression accom-panying cardiac hypertrophy are the result of a multifactorial process and the ob-served changes may therefore be concomitant phenomena. The alteration inventricular gene expression might be an indicator of cardiac hypertrophy and mayresult from a number of different stimuli. We assume that the changes in the rightventricular fibronectin and collagen expression in TF patients could be as a result ofincreased hemodynamic load as it has been often found to be parallel with the in-crease in ventricular mass.19In the case of TF patients, the early increase of collagen and fibronectin deposi-tion in the right ventricle may occur because the original myocardial extracellularmatrix has been expanded and weakened due to the hemodynamic overload. The my-ocardium will compensate until enough new collagen has been produced to restoretensile strength and to resist the distending forces. Our findings in the TF1 and TF2groups fit into this concept as the TF2 shows a more prominent increase in theFIGURE 2. Immunohistochemical localization of fibronectin in right ventricular tissueof patients with tetralogy of Fallot. Micrographs showing human right ventricular tissuestained with rabbit–anti-human fibronectin polyclonal antibodies (as described in Materialsand Methods) from patients undergoing surgery (A) for autograft aortic root replacement(Control), (B) for primary repair for tetralogy of Fallot (TF1), and (C) for secondary surgery(TF2). Bar = 500 µm. (D) Significantly enhanced interstitial fibronectin staining in the TF1group as compared to the respective controls. A semiquantitative analysis of the section wasdone by two independent observers using a staining score ranging from 0 (no staining) to 4(very strong, dark brown, staining) to assess the levels of fibronectin staining (arrows). Val-ues are shown as mean ± SEM.283PETERS et al.: COLLAGEN AND FIBRONECTIN EXPRESSION284 ANNALS NEW YORK ACADEMY OF SCIENCESamount of collagen as compared to TF1. Corrective surgery limits the myocardialoverload and may therefore limit the amount of damage to the myocardial collagenmatrix.6 Our study provides further evidence that myocardial architecture in patientswith TF is altered towards fibrotic state as the expression levels of extracellular ma-trix components like collagens and fibronectin were elevated. However, further re-search at the mRNA level would add to the notion that the gene expression of anumber of proteins involved in fibrosis was transcriptionally altered.SUMMARY AND CONCLUSIONIn the present study the degree of myocardial fibrosis at protein level in patientswith tetralogy of Fallot (TF) was examined by assessing quantitatively the expres-sion of total collagens and fibronectin. We found a significant increase in stainingfor interstitial and total collagens at primary and secondary corrective surgery ascompared to the age-matched control patients. However, immunohistochemical lo-calization of fibronectin revealed significant increase in this protein levels at primarycorrection only. Our results clearly demonstrate an increased amount of myocardialextracellular matrix deposition resulting in tissue fibrosis in TF patients with rightventricular hypertrophy. This vital information could be of great help in assessingthe timing of surgery as well as post-operative prognosis.REFERENCES1. CASTANEDA, A.R. et al. 1994. Cardiac Surgery of Neonate and Infant: 215−234. Saun-ders. Philadelphia. 2. ARAI, M. et al. 1994. Sarcoplasmic reticulum gene expression in cardiac hypertrophyand heart failure. Circ. Res. 74: 555−564.3. VAN BILSEN, M. & K.R. CHIEN. 1993. Growth and hypertrophy of the heart: towards anunderstanding of cardiac specific and inducible gene expression. 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Role of interstitial fibrosis and medial thickening of intramyocardialcoronary arteries. Circ. Res. 69: 107−115.10. CHAPMAN, D. et al. 1990. Regulation of fibrillar collagen types I and III and base-ment membrane type IV collagen gene expression in pressure overloaded rat myo-cardium. Circ. Res. 67: 787−794.11. JALIL, J.E. et al. 1989. Fibrillar collagen and myocardial stiffness in the intact hyper-trophied rat left ventricle. Circ. Res. 64: 1041−1050.285PETERS et al.: COLLAGEN AND FIBRONECTIN EXPRESSION12. HESS, O.M. et al. 1981. Diastolic function and myocardial structure in patients withmyocardial hypertrophy. Special reference to normalized viscoelastic data. Circu-lation 63: 360−371.13. MCLENACHAN, J.M. & H.J. DARGIE. 1990. Ventricular arrhythmias in hypertensiveleft ventricular hypertrophy. Relationship to coronary artery disease, left ventricu-lar dysfunction, and myocardial fibrosis. Am. J. Hypertens. 3: 735−740.14. KAWAI, S. et al. 1984. 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Quantitative analysis of collagens and fibronectin expression in human right ventricular hypertrophy

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Quantitative analysis of collagens and fibronectin expression in human right ventricular hypertrophy

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