J Mol Cell Cardiol

23, 1051-1062 (1991)

In Situ Localization of Tixansfbe Growth Factor & in Porcine Heartz Enhanced Expression after Chronic Coronary Artery Constriction M. Wiinsch,

H. S. Sharma, T. Markert, S. Bernotat-Dauielowski, Kremer, N. Bleese, and W. !3chaper

R. J. Schott,

Kerckhof Clinic and Max Planck Institute, Department of Experimental Cardiolou, Bad Nauheim, Federal Republic of Germany

P.

Benekestr. 2-8, D-6350

(Received 15 August 1990, accepted in revisedfonn 23 April 1991) M. W~NSCH, H. S. SHARMA, T. MARKERT, S. BERNOTAT-DANIELOWSKI, R. J. SCHOTT, P. KREMER, N. BLEESE. AND W. SCHAPER. In Sifu Localization of Transforming Growth Factor fit in Porcine Heart: Enhanced Expression after Chronic Coronary Artery Constriction. Joumd of Molecular and Cellular Cardiolo~ (1991) 23, 1051-1062. We investigated the expression of transforming growth factor beta 1 (TGF-f31), a polypeptide differentiation factor probably associated with angiogenic properties in chronically hypoperfused heart tissue. A slowly swelling ameroid constrictor was implanted around the coronary circumflex artery (CX) of young domestic pigs. Two to three weeks after, significant CX stenosis of more than 90% and coronary collateralization could be demonstrated angiographically. The CX dependent experimental myocardial tissue (E) was investigated, with the LAD dependent area of the same pig serving as a control (C). We found significantly enhanced TGF-/31 mRNA expression by northern blot hybridization in the experimental myocardium (E) of those pigs with demonstrable coronary collaterals in the absence of a major myocardial infarction. The presence of TGF-01 protein could be demonstrated quantitatively in extracts of the experimental and the control area by immunoblot analysis. By in sifu techniques, TGF-01 mRNA and protein could be localized predominantly in cardiac myocytes. We conclude that one adaptive mechanism ofthe pig heart in chronic coronary artery constriction is the enhanced expression of TGF-01. Cardiac myocytes are a major source of TGF-/31. The observed coronary collateralization could be mediated - at least in part - by the angiogenic properties of TGF-01 KEY WORDS:

TGF-PI;

Angiogenesis;

Coronary

constriction;

Introduction Coronary artery collaterals develop in response to slowly progressive coronary artery stenosis during atherosclerotic heart disease (Fulton et al., 1963, Shaper et al., 1971 and 1988). The protection of myocardial tissue and function in the case of a sudden critical stenosis depends in large part on the extent of performed or newly developed coronary collateral networks (Schaper et al. 1988, Epstein et al., 1989). Polypeptide growth factors play an important role in the formation of new blood vessels (angiogenesis) during wound healing (Folkman and Klagsbrun, 1987; Deuel, 1987; Risau and Zerwes, 1989) and tumor growth. Earlier studies from our group suggest ‘primary biochemical transmitters of growth’, i.e. angiogenic growth factors, to be responsible for the development of coronary collaterals, too (Pasyk et al., 1982). All correspondence should be addressed D-6350 Bad Nauheim, Federal Republic 0022-2828/91/091051

+ 12 $03,00/O

to M. Wiinsch, of Germany.

Swine

heart;

in situ techniques.

Potent angiogenit growth factors, such as fibroblast growth factors, FGFs (Quinckler et al., 1988) and transforming growth factor /3i, TGF-/3 (Thompson et al., 1988, Eghbali et al., 1989), have been demonstrated in the myocardium of various mammals. TGF-01, a 25.000 d homodimeric polypeptide was first characterized by its ability to induce anchorage-independent growth of non-neoplastic fibroblasts (Moses et al. 1981, Roberts et al., 1981, Anzano et al. 1982). It has been found to elicit a potent angiogenic reaction when injected subcutaneously into newborn mice (Roberts et al., 1986) or when applied locally in wound healing experiments (Sporn et al., 1983, Mustoe et al., 1987, Pierce et al., 1989). We investigated the presence of TGF-01 in a porcine model of chronic coronary stenosis induced by ameroid constriction of the left cirKerckhoff-Clinik,

Dept.

of Cardiac

Surgery,

0 1991 Academic

Benekestr.

Press

2-8,

Limited

1052

M. Wiinsch

cumflex coronary artery (CX). An enhanced expression of TGF-Pl mRNA in the experimental, CX dependent tissue was found by Northern hybridization. TGF-/31 protein and mRNA could be localized by in situ techniques to myocardial cells around damaged tissue but not to sites of tissue repair with their reactive inflammatory infiltrate.

Materials Animal

and Methods

surgery and tissue preparation

male domestic pigs (German Twenty Landrace), two month of age with a mean weight of 17.4kg (S.D. 1.25) were used for this study. The pigs were intubated endotracheally and ventilated with a mixture of room air and oxygen under general anaesthesia with nembutal and ketanest. A lateral thoractomy was performed and an ameroid constrictor was implanted around the left circumflex coronary artery (CX) as described earlier (Gorge et al., 1989). Two to three weeks later, coronary arteriographic studies were done via the carotid artery. The degree of stenosis, as well collateralization present were as, any documented. In the absence of significant stenosis coronary angiography was repeated one week later. The pigs were sacrificed and the heart was removed and quickly rinsed in ice cold normal saline. Tissue samples of the CX area and of the normally perfused anterior wall and septum were exicsed selectively, frozen immediately in liquid nitrogen or isopentane and stored at - 80°C until analysis.

RNA preparation

and Northern blot analysis

Frozen tissue was homogenized and total RNA was extracted under 6M guanidiniumisothiocyanate (Chirgwin et al. 1979). RNA concentrations were defined by measuring the optical density (OD) at 260nm on an UV spectrophotometer (Ultrospec of Pharmacia LKB Biotechnology, Uppsala, Sweden). Equal amounts of total RNA were electrophoretically separated on a 1% agarose gel containing 2% formaldehyde and ethidium bromide and osmotically transferred to a Hybond nylon (Amersham Buchler, membrane Braunschweig, FRG). To control for quality

et al. and quantity of the transfer, the nylon membrane was fotographed on UV-light at 260nm. A 1050bp human cDNA probe encoding TGF-01 (Derynck et al., 1985) was labeled by means of a multiprime labeling kit (Amersham) and oJ2P dCTP to 10gcpm/mg DNA. Hybridization was performed at 42°C containing 50% deionized in a buffer formamide, 1M NaCl, 0.2% polyvinyl pyrolidone, 0.2% BSA, 0.2% ficol, 0.1% sodium pyrophosphate, 1% SDS, 50 mM Tris (pH 7.5), 10% dextransulphate and 50 mg/ml denatured salmon sperm DNA. The nylon membrane was washed 2 x 5 min at 20°C in 2 x SSC containing 0.1% SDS, 30min at 6O’C in 1 x SSC (0,l % SDS), 30 min at 60°C in 0,l x SSC (O,l% SDS), and 2 x 5 min at 2O’C in 0,l x SSC without SDS. Then the membrane was exposed to a Kodak XAR-film for 1 to 3 days at - 7O’C.

Immunohistochemical

studies

Five pm frozen sections of myocardial tissue were investigated immunohistochemically using three different antibodies: (1) polyclonal anti TGF-01 antibody raised in rabbits by the use of synthetic peptides corresponding to the 29 N-terminal amino acids of human TGF-Pl (gift from Van den Eijnden-Van Raaij, 1988); (2) non neutralizing and (3) neutralizing polyclonal anti TGF-01 antibodies prepared in rabbits by injecting TGF-01 purified from porcine platelets (British Biotechnology Inc., Oxford, U.K.). The following protocol was applied: permeabilization by 100% acetone at - 2O“C; blocking of non-specific protein binding by incubation with 10% BSA or 10% goat serum (45 min at 37W); incubation with 5 % or 10% of one of the anti-TGF-01 antibodies in PBS buffer containing 10% BSA or fetal calf serum (60min at 37’C); incubation with 2% affinity purified biotin conjugated goat anti-rabbit IgG (60 min, 37’C, British Biotechnology); detection and staining of the antibody complexes by streptavidine-fluoresceine (45 min, 37°C); counterstaining of nuclei by orange fluorescing propidium iodide. Controls were done simultaneously by replacing either the first antiTGF-/31 antibody or the second anti-rabbit antibody by 10 % BSA, 10 % fetal calf serum or 1% rabbit serum.

Transformin

g Growth

Factor

Bl

Protein extraction and Western blot analysis Total protein was extracted from homogenized myocardial tissue in a buffer containing 62.5 mM Tris, 2 % SDS, 2 mM phenylmethylsulfonylfluoride, 15% sucrose and 2 mM EDTA at a pH of 6.8. Proteins were electrophoretically separated on a 12% polyacrylamide gel containing 0.1% SDS. Proteins were either silver stained or electroblotted to a 0.45 firn nitrocellulose membrane (Schleicher und Schuell, Dassel, FRG). TGF-/31 on the filter was detected as follows: Incubation of the filter with PBS buffer containing 0.1% NsNa for 1 h; blocking of non-specific binding by incubation for 3 to 4 h in a blotting solution of 5% milk powder, 50 mM Tris (pH 7.8) 2 mM CaCl,, 0.05% Triton X-100, 0.01% antifoam; incubation with polyclonal rabbit and anti-TGF-/31 antibody (van den Eijndenvan Raaij et al., 1988) in a dilution of 1: 100 in blotting solution of 1: 100 in blotting solution for 2.5 h at 20°C; incubation with 1:200 goat anti-rabbit IgG coupled to alkaline phosphatase for 2 h at 20% (Amersham); incubation with p-nitrobluetetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate ptoluidine salt (BCIP) in alkaline buffer (pH 9.4) for color development. TABLE

1. Characteristics

of the pigs Days of constrictor implantation

in Chronic

Myocardial

Ischemia

1053

In situ hybridization The TGF-01 antisense riboprobe was generated from a 1050 bp human cDNA (Derynck et al., 1985), inserted in the EcorRl site of the pSP64 vector in the antisense orientation with respect to the SP6 promotor by the use of SP6 RNA polymerase (Promega Inc., Madison, USA). Riboprobes for the controls were synthesized from a DNA fragment containing a sequence common to the (Y and /3 subunits of the rat myosin heavy chain gene, MHC, (K. Schwartz, personnal communication), cloned into the Bluescribe vector (Stratagene, La Jolla, CA., USA). Sense and antisense probes were synthesized by using T3 RNA polymerase (Bijhringer Mannheim, FRG) and T7 RNA polymerase (Promega Inc.), respectively. Riboprobes were synthesized in the presence of 35S-(w-UTP (370 MBq/ml, Amersham) to a specific activity of 108cpm/~g of RNA and partially hydrolyzed to yield an average fragment length of 100 to 150 bp (Cox et al., 1984). Sample preparation and hybridization were done following in principle the protocol of Hogan (1986) and others (Brahic and Haase 1978, Simmons et al., 1989). In brief, frozen sections of 5mm were lyophilyzed on slides

investigated Coronary stenosis

Pig Nr.

Weight (kg)

Pig

1

17

14

100

Pig

2

17

16

100

Pig

3

19.5

18

100

Pig Pig

4 5

15.9 16.3

18 14

75 95

Pig

6

18.4

16

90

Pig

7

18.6

15

100

Pig Pig

8 9

16.9 15.8

15 13

75 100

Pig 10 Pig 11

17.8 18.5

16 16

95 95

(%I

Left ventriculogram inf.-lat. akinesia inf.-lat hypokinsia lat. wall akinesia

normal inf. lat. hypokinesia normal lat. hupokinesia normal inf. lat akinesia normal normal

Collaterahzation

Infarction

none

major

from

RCA

spotty

from prox. CX faintly from RCA none from LAD

spotty, microscars

from

LAD

spotty

from

LAD

major

none from

RCA

none major

none none

none none

none none

1054

M.

Wiinscb

pretreated with 3-aminopropyltriethoxysilane (Van Prooijen-Knegt et al., 1982), fixed in 4% paraformaldehyde for 20mins at 20°C and dried. The hybridization buffer contained 40% deionized formamide, 0.6M NaCl, 5 mM EDTA, 0.02% Ficoll 400, 0.02% polyvinylpyrolidone , 16.8 mM NaH2P04, 33 mM Na2HP04, 0.02 % BSA and 0.1% degraded salmon sperm DNA. Hybridization was done at 65OC over night. The slides were washed three times at room temperature in a buffer containing 50% formamide, 1 x SSC and 0.1% SDS at 37OC and two times at 65OC. The tissue sections were digested by RNAse A (1 mg/ml) and again rinsed in washing buffer at 65OC for 4 hours. The slides were dipped in Amersham LM-1 photoemulsion and exposed for 7 to 14 days at 4’C. After developing the slides were counterstained with toluidine blue.

Results Two the three weeks after placement of the ameroid constrictor around the circumflex

et

al.

artery of pigs, the degree of constriction was investigated by coronary angiography. In 11 out of 20 pigs significant stenosis or occlusion was demonstrated (Table 1). Macroscopically demarcated myocardial infarction was seen in three of the pigs [I, 7, 91. Functionally significant wall motion abnormalities could be demonstrated by ventriculograms in these three pigs and in three additional ones (2, 3 and 5) without major infarction but with histologically visible spotty necroses. Coronary artery collaterals, originating proximally or distally from the stenosis were visible in pigs 2, 3, 5, 6, 7 and 9. Figure 1 shows the results of a representative Northern blot hybridization. The TGF+l cDNA probe was hybridized specifically to two mRNA bands of 2.4 and 3.5 kb in all samples investigated. In pig 1 (major infarction and no collateralization) there is an intense signal for TGF-01 mRNA with no difference between experimental and control tissue. Pigs 2 and 3 (visible collateralization and only spotty infarction) have a significantly higher

(a) Pip

Er

28s

3.5

hb

2.4

hb

A

I

M

A

CO

EX

co

Ex

A CO

En

CO

RNA

FIGURE 1. Northern hybridization analysis of TGF-01 expression in experimental and control myocardial tissue. Twenty mg of extracted total RNA per lane were transferred to a filter membrane and photographed at 560 nm UV light. The filter was subjected to Northern blotting with a 32P labeled TGF-/31 cDNA probe. The autoradiography shows a 2,4APD,, kb and a 3.5 kb mRNA signal in all lanes. Signals from experimental tissue (E) are enhanced compared to control (C) in pigs 2 and 3. At the right side the positions of the 28s and the 18s ribosomal RNAs is indicated. At the bottom the 28 S ribosomal RNA bands of each lane are shown photographed at UV light. They indicate an almost equal transfer of total RNA to the filter.

Transforming

Growth

Factor

Bl

TGF-&-e

-21.5

FIGURE 2. Immunoblot analysis of TGF-01 in normal and experimental porcine myocardium. Immunoblot of a 12.5 ‘$6 SDS-polyacrylamide gel electrophoreses (non reducing conditions) after incubation with a polyclonal anti TGF-01 antibody (prepared in rabbit). Detection of specific binding was done by anti rabbit antibody coupled to alkaline photpbatase. (1) 50ng of purified TGF-@I; (2) 35mg protein extract from ischemic and (3) 35 mg from normal porcine myocardium. At the right side the corresponding positions of protein molecular weight markers are noted.

expression of TGF-01 specific mRNA in the experimental region. Pig 4 without a hemodynamically significant stenosis shows an obviously weaker mRNA-signal than pig 1, but no difference between the two regions. The lower panel of Figure 1 demonstrates the excised 28s RNA bands of each lane on the membrane, stained by ethidium nylon bromide and fotographed on UV-light (260 nm). Immunoblot analysis with a polyclonal anti TGF-01 antibody shows specific staining of a protein band of 25.000d (Fig. 2, lanes 2 and 3), corresponding to the published size of TGF-01 (lane 1). No significant difference in antibody binding was evident between control (lane 2) and experimental (lane 3) protein extracts. Figure 3 shows in situ mRNA

in Chronic

Myocardial

Ischemia

1055

hybridization results using a TGF-/31 specific antisense riboprobe. Figure 3a depicts a toluidine blue stained section demonstrating the typical appearance of the border between intact myocardium (upper part of the figure) and a focal necrosis. Figure 3b shows a darkfield microphotography of the same section. Dense silver granulae, indicating hybridization, are detected in intact myocardium (upper part of the figure and an insula at the left bottom). Remodelling fibrotic tissue shows only weak reactivity. A myosin heavy chain sense probe served as the control for specificity of the hybridization technique used and showed only background staining (3~). The corresponding myosin heavy chain antisense probe showed dense staining throughout the entire myocardium [Fig. 3(d)]. Five pm frozen sections of myocardial experimental tissue were stained by immunofluorescence technique using three different polyclonal anti TGF-@l antibodies. The results shown in Figure 4 were obtained by using a polyclonal TGF-fll antibody, raised against aminoacids l-29 of the mature human protein (Van den Eijnden et al., 1988). Figure 4(a) shows an unstained section through the experimental tissue by phase contrast technique: A small necrosis centrally (arrow), surrounded by histologically normal appearing myocardium. Figure 4(b) shows a light green stain of the intact cardiac myocytes. In the scar tissue the intense orange staining of nuclei indicates dense infiltration of inflammatory cells. A TGF-01 specific green stain is absent. Figure 4(c) shows a negative control, where 10% fetal calf serum was substituted for the first anti TGF-fll antibody. In other experiments 10% bovine serum albumin and 1% rabbit serum were substituted for the first antibody as well as 10% BSA for the second one (data not shown). Platelets of a blood smear served as a positive control and could be stained clearly by the antibody applied (not shown). Using a polyclonal, not neutralizing antibody prepared by the injection of complete porcine TGF-01 in rabbits (BRL), we could demonstrate distinct perinuclear staining of a few myocytes (not shown). Using a neutralizing antibody of the same manufacturer we saw a fine pericellular staining pattern including the T-tubuli system, reminiscent of the staining pattern of the

1056

FIG 5mm tissue result intact show probe antise

M. Wiinsch

et al.

;URE 3. In situ hybridization of TGF-@l mRNA in sections of experimental porcine myocardium. (a) she IWS a frozen section through the experimental tissue stained by toluidine blue. At the top histologically intact myoca rdial is shown, at the bottom scar tissue consisting of connective tissue and a variety of infiltrating inflammatory cells ing from a micronecrosis; (b) Darkfield microphotography of the same section showing dense silver granulae i n the myocardium indicating TGF-fll specific hybridization. The scar tissue shows only background activity; (c) an d (4 i darkfield microphotograph& of control sections hybridized with myosin heavy chain sense and antisense ribos, respectively: (c) Minimal background staining of the sense probe; (d) intense staining of the correspon .ding ‘rise probe. Note the spared area around the cross-sectioned coronary vessels. (ZOO x ).

Transform&

g Growth

Factor

Bl in Chronic

Myocardid

Ischemia

F ‘IGURE 4.Immunohistochemica1 staining of TGF-@I protein in sections of experimental myocardium. (a) shol ws a microphotography of a 5 mm frozen section of the experimental myocardial tissue. Histologically in [tact pha .se contrast cells, most of them cross sectioned, surround a small area of damaged tissue (arrows); (b) mYf ocardial the II”1 nunofluorescence microphotography of the same site shows light green staining of cardiac myocytes bordering dan naged area. The scar tissue is free of stain. Nuclei are counterstained orange by propidium iodide; (c) In a sin iilar :ion 10% fetal calf serum was substituted for the first antibody as a negative control. No green stain is visible. (a al ndb 200 1x , c 170 x ).

1058

M. WGnsch et al.

GURE 5. (a) HE stain of a strand of porcine Jing large, nucleated cells with the characteristic specific anti-TGF-01 antibodies. (a) 250 x ; (b) 200

purkinje cells; (b) I mmunohistochemical perinuclear halo of Purkinje cells. x ).

structural protein laminin (Shaper J. unpublished data). Interestingly, in some sections of control as well as experimental tissue we found a cluster of nucleated cells slightly larger than cardiac myocytes with a perinuclear halo. They could be identified morphologically as porcine Purkinje cells [Fig. 5(a)]. These cells were stained intensely positive by TGF-/31 specific antibodies [Fig. 5(b)] as well as, by anti desmin antibodies (data not shown). Discussion TGF+31 and its mRNA have been found in heart tissue by several investigators. It has been localized in the developing cardiovascular system of the mouse embryo (Heine et al. 1987) and after acute myocardial infarction due to coronary artery ligation in rats (Thompson et al., 1988). To study TGF-/31 expression during coronary vessel collateralization we investigated porcine hearts after

They

stain of the are brightly

same aIrea, stainel d by

chronic coronary constriction. By Northern hybridization we found a significantly enhanced expression of TGF-/31 mRNA in the ischemic as compared to the control tissue in those pigs with critical stenosis but without a major infarction (Fig. 1, pigs 2 and 3). Even without a more sophisticated quantification the difference between these lanes is apparent. Equal loading and transfer of RNA is demonstrated by the photograph of the 28s ribosomal RNA band of each lane (Fig. 1 lower panel). The 2.4 kb mRNA signal detected corresponds to the length of the published TGF-01 mRNA (Derynck et al. 1985, Derynck and Rhee 1987). An additional TGF-@l specific mRNA species of 3.5 kb is present and is probably generated by the selection of an alternate polyadenylation site (Kondaiah et al., 1988; Van Obberghen et al. 1987). Chronic myocardial ischemia induces an upregulation of TGF-/31 mRNA in our animals as intense as major infarction (Fig. 1, Pig 1).

Transforming

Growth

Factor

Bl

Extracted total tissue RNA averages RNA originating from necroses, regenerating tissue and bordering myocardium. Northern blot analysis cannot well differentiate between contributing tissue components. It is therefore understandable, that the cellular source of cardiac TGF-Pl is controversial in the literature: Thompson et al. (1988) found TGF-01 mRNA and protein in cardiac myocytes whereas Eghbali (1989) could demonstrate it only in the non-myocyte fractions of cardiac tissue. Using in situ mRNA hybridization, we found TGF-/31 specific activity clearly distributed in histologically intact myocardium (Fig. 3). Myosin heavy chain cDNA sense and antisense probes were hybridized in parallel as controls. Myosin heavy chain is the ideal control because of its abundance and specificity in cardiac tissue. The sense probe showed only weak background activity scattered throughout the section which indicates only minimal unspecific binding. We could demonstrate TGF-01 specific staining in cardiac myocytes also by immunodetection of TGF+?l protein (Fig. 4). Connective tissue, especially around blood vessels, did not show more than background activity. With other TGF-@l specific antibodies we found different staining patterns of cardiac myocytes, depending on the antibody used. Recently, a similar variation in the staining pattern of different TGF-/31 antibodies has been described by others (Flanders et al. 1989). We believe, that immunohistochemical detection of TGF-01 in tissue reflects the site of its production only poorly. Different antibodies bind TGF-01 selectively in its various functional environments such as cytoplasm, cell membranes and interstitiel tissue, according to the individual subset of antigenic determinants exposed. One basis for this variability may be the association of TGF-01 or its latent form with other proteins, which can mask its antigenic determinants. Such an associate has been described for human TGF-/31 as a glycoprotein of 135kd distinct from al-macroglobulin (Wakefield et al., 1988). Interestingly, we do not see a difference of TGF-/31 mRNA expression between control and experimental tissue in the pigs with complete CX occlusion and a major infarction

in Chronic

Myocardisl

Ischemia

1059

(pig 1; pigs 7 and 9 not shown). Probably, coronary artery occlusion occurred suddenly after constrictor implantation. The dependent myocardium had no time for adaptive tissue responses and underwent almost complete necrosis. Although TGF-01 plays an important role in tissue repair (Sporn et al., 1983, Mustoe et al., 1987), and is secreted by fibroblasts (Roberts et al., 1981, Flanders et al., 1989) and activated macrophages (Assoian et al., 1987), after acute myocardial infarction it does not seem to play a significant role. This is another indicator that intact myocardium, and not regenerating connective tissue, is the major source of cardiac TGF-fll. The presence of TGF-01 protein in our model was demonstrated by Western immunoblot analysis (Fig. 2). We found no difference in the intensity of the signal between control and experimental tissue extracts. However, the alkaline phosphatase antibody detection system may not allow fine quantification. An equal amount of mature TGF-01 protein in the experimental area may also be due to’ a higher protein turnover. TGF-01 receptors have been found in a broad spectrum of cell types including vascular endothelial cells (Wakefield et al., 1987, Massague and Like 1985, Cheifetz and Massague 1989). Upregulation of TGF-01 receptors could increase internalization and degradation of TGF-/31 resulting in a steady state protein level in the presence of substantially increased mRNA transcription. An initial rise of TGF+l protein may also have been missed, since we investigated the pigs when maximum stenosis has been present for some days. Moreover, a poor correlation between TGF-/31 mRNA levels and the rate of TGF-01 secreted into culture supernatants has been described (Kehrl et al., 1986). An interesting additional finding was the detection of an intensely stained cluster of large nucleated cells with the histologic appearance of Purkinje cells in sections of the control, as well as, experimental myocardium [Fig. 5(a) and (b)]. Cells of the cardiac conduction system have not been described before to excrete or bind TGF-01. Possibly, TGF-01 and other growth factors in these highly organized cells have an important role in the establishment and maintenance of cardiac muscle organization. In earlier experiments

1060

et

M. Wiinsch

with chronic constriction of porcine coronary arteries, blood vessels near the conduction system were the only macroscopically visible collaterals (Schaper et al., 1967). Cardiac myocytes are especially sensitive to lack of oxygen because of their intense aerobic metabolism. We conclude, that one of their adaptive reactions to chronic ischemia is the enhanced expression of TGF-fll . TGF-01, in conjunction with other angiogenic growth factors, especially the FGFs (Quinckler et al. 1988, Sharma et al. 1989) might be a major determinant for the observed ingrowth of capillaries and macroscopic coronary collateral vessels in the case of chronic coronary artery stenosis.

Limitations

of the method

ischemia induced myocardial To study collateral vessel growth and its possible molecular basis, a model was sought which would produce coronary artery stenosis sufficiently gradual enough to allow maximal adaptive tissue response without ischemic necrosis and the resultant confounding repair mechanisms. The ameroid model is a well established method to induce coronary artery stenosis slowly by hygroscopic swelling (Litvak et al., 1957, Schaper et al., 1966). As was demonstrated recently by Gorge (1989) in the pig heart, coronary blood flow was reduced to about 20% of normal 2-4 weeks after implantation of an ameroid constrictor and collateral flow rose to 60 ‘$ of maximal normal flow. Numerous small vessels developed throughout the area at risk in a dense ‘blush’-like fashion. For these characteristics the pig model approximates the situation in the human heart better than other animal models, having either a large scale of naturally

al.

occurring collaterals (dog) or no possibility for collateral opening and formation (rat) (Schaper et al., 1971, 1987). Nevertheless, the ameroid dependent stenosis, as a biological process, differs widely. Neither complete occlusion nor minor stenosis can be supposed to induce optimal collateral vessel growth. Therefore, we performed angiography studies to define the individual grade of stenosis. Eleven pigs out of a series of 20 were eligible for further molecular biological investigation (Table 1). They represent the spectrum of interesting stenoses and their pathophysiologic consequences. Pigs 2, 3, 5 and 6 with high grade coronary stenosis and collateralization but without a major infarction may best simulate the situation of humans with chronic coronary heart disease before myocardial infarction (reviewed by Epstein et al., 1989). Nine pigs either died before the end of the protocol (3 pigs) or did not develop high grade stenosis (6 pigs). Therefore, the number of pigs investigated is limited. However, it was not the purpose of our study to do a statistical analysis on the pathophysiologic range of TGF-Pl in the pig heart. The positive finding of TGF-@l in all hearts investigated and the demonstration of significant upregulation in four pigs supports our hypothesis sufficiently.

Acknowledgement We thank Drs J. Schaper, K. Fleischmann, T. Brand and S. Rohman for many helpful discussions and for critically reading the manuscript. We thank E. Neubauer and R. Froede for technical assistance. The cDNA probe for TGF-01 was kindly provided by Dr R. Derynck. The anti TGF-01 antibody and TGF-01 protein were kind gifts from Dr van den Eijnden-van Raaij.

References ANZANO, MA, ROBERTS A, MEYERS C, KOMORIYA L, LAMB L, SMITH J, SPORN MB (1982). Synergistic interactions of two classes of Transforming Growth Factors from murine sarcoma cells. Cancer Res 42, 4776-78. ASSOIAN RK, KOMORIYA A, MEYERS CA, MILLER DM, SPORN MB (1983). TGF-@ in human platelets. J Biol Chem 258, 7155-7160. ASSOIAN RK, FLEURDELYS BE, STEVENSON HC, MILLER PJ, MADTES DK, RAINES EW, Ross R, SPORN MB (1971). Expression and secretion of type @ TGF by activated human macrophages. Proc Nat1 Acad Sci USA 84, 6020-6024. BRAHIC M, HAASE AT (1978). Detection of viral sequences of low reiteration freqauency by in situ hybridization. Proc Nat1 Acad Sci USA 75, 5125-6129. CHEIFETZ S, MASSAGUE J (1989). T ransforming Growth Factor /3 receptor proteoglycan J Biol Chem 264, 12025-12028. CHIRCWIN JM, PRZYBYLA AE, MCDONALD RJ, RULER WJ (1979). Isolation of biochemically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18, 5294-99.

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In situ localization of transforming growth factor beta 1 in porcine heart: enhanced expression after chronic coronary artery constriction.

We investigated the expression of transforming growth factor beta 1 (TGF-beta 1), a polypeptide differentiation factor probably associated with angiog...
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