Skip Navigation

Cardiovascular Research 2002 55(1):113-121; doi:10.1016/S0008-6363(02)00340-1
© 2002 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Basso, C.
Right arrow Articles by Iliceto, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Basso, C.
Right arrow Articles by Iliceto, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 2002, European Society of Cardiology

Endothelin A-receptor antagonist administration immediately after experimental myocardial infarction with reperfusion does not affect scar healing in dogs

Cristina Bassoa, Gaetano Thienea,*, Mila Della Barberaa, Annalisa Angelinia, Michael Kirchengastb,c and Sabino Ilicetod

aInstitute of Pathological Anatomy, University of Padua Medical School, Via A. Gabelli 61, 35121 Padova, Italy
bInstitute of Pharmacology and Toxicology, University of Heidelberg Medical School, Mannheim, Germany
cCardion AG, Erkrath, Germany
dDivision of Cardiology, University of Padua Medical School, Padova, Italy

cardpath{at}unipd.it

* Corresponding author. Tel.: +39-049-827-2283; fax: +39-049-827-2284

Received 7 November 2001; accepted 27 February 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions and clinical...
 References
 
Objective: Endothelin (ET) receptor antagonists have been reported to reduce both infarct size and no-reflow phenomenon; however, in rat models their effect on the healing process after myocardial infarction (MI) is controversial. The study aimed to evaluate the effect of early administration of the ETA receptor antagonist darusentan on scar healing in an ischemia-reperfusion model in dogs. Methods: Thirty male mongrel dogs surviving 180 min left anterior descending coronary artery balloon occlusion were randomised to: darusentan i.v. bolus—5 mg/kg 5 min before reperfusion—(group I); darusentan i.v. bolus+chronic oral—10 mg/kg/day—(group II); saline (group III). Five age-matched dogs served as controls (group IV). At 6 weeks weight, volume, mass/volume, wall thickness, thinning ratio and expansion index were assessed in the explanted hearts. Infarct size and scar area tissue composition were evaluated by computerized histomorphometry. Cellularity, vessels and TGFβ in the scar area were scored by immunohistochemistry. Results: 24 dogs (80%; 7 group I, 8 group II, 9 group III) developed an anterior MI, transmural in 15 and subendocardial in 9, mean size 11.5±4% of left ventricular area and 37±9% of left ventricular endocardial circumference. MIs were homogeneously distributed among the three groups regarding either infarct size or transmural extent. No differences were found in the three MI groups regarding thinning ratio, expansion index and scar area tissue characterization. Percent scar collagen content (37±17 vs. 53±20 vs. 46±14), myofibroblasts (1.2 vs. 1.3 vs. 1.4), macrophages (1.2±0.5 vs. 1.3±0.5 vs. 1.4±0.5), neovessels (2.8±0.4 vs. 2.6±0.5 vs. 2.9±0.3) and TGFβ score (2 vs. 2.25 vs. 2.11) were not significantly different. Conclusions: Early administration of the ETA receptor antagonist darusentan does not affect the scar healing process at 6 weeks after experimental MI with reperfusion in dogs.

KEYWORDS Endothelins; Fibrosis; Histo(patho)logy; Infarction; Reperfusion


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions and clinical...
 References
 
Endothelin (ET) is a 21 amino acid peptide with a powerful vasoconstrictor effect in both conductive and resistive arteries [1,2]. Plasma and tissue levels of ET increase during ischemia and reperfusion, suggesting a pathophysiological role in the genesis of post-ischemic microvascular and myocardial damage [3–5]. The administration of ET receptor-antagonists before coronary occlusion has been shown to reduce infarct size [6,7]. In a canine model of 90 min coronary occlusion followed by 3 h reperfusion, we have shown that the non-peptide ETA receptor antagonist darusentan, when administered intravenously 5 min before reperfusion, reduces not only infarct size but also no-reflow phenomenon within the risk area [8].

Although chronic treatment with ETA receptor antagonists has been demonstrated to improve ventricular remodeling and survival in rats when started 10 days post myocardial infarction (MI) [9,10], other data suggest that onset of treatment within 24 h post-MI may lead to impaired scar healing, left ventricular (LV) dilatation and dysfunction [11,12].

The aim of the present study was to evaluate by a histomorphometric and immunohistochemical analysis the effect of acute intravenous administration of darusentan either alone or followed by chronic oral treatment, on the scar healing process 6 weeks after coronary occlusion and subsequent reperfusion in dogs.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions and clinical...
 References
 
2.1 Experimental protocol
The protocol was approved by the local ethics committee (Tierschutzkomission der Bezirksregierung Rheinhessen-Pfalz) and conformed to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory and Animal Resources, Commission of Life Sciences, National Research Council—USDHHS, PHS, NIH publication No. 85-23, revised 1985).

Thirty-eight male mongrel dogs (Hazleton, USA) weighing 11.5 to 15.0 kg were used. For coronary artery occlusion animals were anesthetized by an intravenous injection of 30 mg kg–1 pentobarbital sodium followed by continuous infusion of 3 mg kg–1 h–1. After intubation, the animals were artificially ventilated with a mixture of 25% O2 and 75% N2O. Body temperature was maintained at 38.5±0.5 °C with a temperature-controlled heating table.

The left anterior descending coronary artery (LAD) was occluded using a percutaneous transluminal coronary angioplasty (PTCA) balloon catheter. The catheter was introduced via the left carotid artery; a standard PTCA guide catheter (Judkin left, powerbase 8F, ACS; mandrin=softguide, ACS) was advanced via the aortic root into the LAD under X-ray guidance. The balloon catheter (RX Elipse 0.014, ACS) was positioned in the middle portion of the LAD using a 0.014 in. PTCA guide wire (Galeo, Biotronic) (1 in.=2.54 cm). After X-ray control of its position, the balloon catheter was expanded by inflating to 0.5 atm for 180 min (1 atm=101 325 Pa). After deflating and reperfusion, the catheter was removed, the incision in the carotid artery sewn and the animals returned to the animal housing and placed underneath a heating lamp.

Dogs surviving coronary occlusion were randomized to: (1) darusentan bolus acute (group I); (2) darusentan bolus acute followed by chronic oral administration (group II), (3) saline (group III). Five age-matched dogs who did not undergo LAD occlusion served as controls (group IV). After 6 weeks dogs were re-anesthetized, ventilated and thoracotomized. The dogs were sacrificed by an overdose of pentobarbital and the hearts explanted to perform post-mortem analysis.

2.2 Drug
Darusentan=LU 135252: (+)-(S)-2-(4,6-dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-dyphenyl-propionic acid (Knoll, Ludwigshafen, Germany) is a selective endothelin ETA receptor antagonist [13]. The medication half-time in dogs is 12–14 h. For intravenous use the compound was dissolved in aqueous solution containing 0.1 M NaOH, buffered by addition of HCl to a pH of 7.0±0.1 and administered 5 min before reperfusion at 5 mg/kg. For oral application, the compound was administered to dogs in a soft gelatine capsule once daily via gavage at 10 mg/kg. We adopted a lower dose, in comparison with previous experiments dealing with ET receptor antagonists, since recent studies demonstrated the drug efficacy at a daily dosage of 5–10 mg/kg both in the treatment of congestive heart failure [14] and in blood pressure lowering [15].

2.3 Pathology protocol
LV volume was measured by ligating the aorta at the sino-tubular junction, opening the left atrium, filling the LV cavity with saline to the mitral level, and measuring the total amount of saline needed. Subsequently the hearts were perfused for 20 min with 10% phosphate-buffered formaldehyde at 100 mmHg (1 mmHg=133.322 Pa). The specimens were put in a jar containing 10% buffered formaldehyde until analyzed in a blinded fashion.

Each specimen was weighed, and longitudinal and transverse diameter were measured. Four transverse 5-mm slices of the LV, from cardiac apex to base, were cut and photographed. Using an image analyser system (TV camera 3CCD JVC; microscope Olympus; scanner Duoscan T 1200 Agfa, personal computer with Image-Pro Plus software version 4.0 by Media Cybernetics, Silver Spring, MD, USA) the mass/volume ratio (M/V) was assessed on each transverse slice by dividing the LV area (resulting from the outline of the entire slice of the LV measured by digital planimetry less the LV cavity area measured identically) by LV cavity area. The average M/V value of four slices was used.

The transverse slices of the LV were embedded in paraffin and three 7-µm thick sections were cut from each paraffin block, and stained with hematoxylin–eosin, Mallory triple stain modified by the Heidenhain technique, and Sirius Red. Five additional sections were cut to perform immunohistochemistry investigation (see below).

The infarct was defined as subendocardial when a rim of undamaged myocardium was interposed between the scar area and the epicardium, and as transmural when the scarring tissue extending from the endocardium was reaching the epicardium. Scar size (scar extent) was assessed by tracing the outline of replacement-type fibrosis in each transverse slice and by calculating the area by digital planimetry. Scar size was expressed as percentage of LV myocardial area. The endocardial circumference of scar tissue and of the entire LV were determined to calculate scar size, expressed as endocardial circumference of the scar tissue divided by LV endocardial circumference. Thicknesses of the infarct and non-infarct ventricular ring along radials at 45° angular intervals were measured, by excluding the trabeculae and papillary muscles. The thinning ratio and expansion index were calculated using a modification of the method by Eaton and Bulkley [16]. The transverse slice containing the greatest percent of scar tissue was selected for each heart, and the lengths of the endocardial margins for the anterior and posterior segments as defined by the point of intersection of the septal bisector (i.e., line drawn from the midpoint of the septum between anterior and posterior junctions with the right ventricle perpendicular to the septum, to meet the free wall) were measured. Thus, the expansion index was calculated as the endocardial length of the scar-containing anterior segment/endocardial length of the non-infarcted posterior segment; and the thinning ratio as the average thickness of the scar-containing anterior segment/average thickness of non-infarcted posterior segment.

2.4 Scar healing process analysis
For tissue characterization, three different fields using a fourfold magnification were examined on sections stained by Heidenhain trichrome and the average values were calculated. Fibrosis was quantified by computerized morphometry as the sum of all connective tissue divided by the total corresponding scar area. The same procedure was applied to measure percent of viable myocardium and of remaining tissue (loose connective, vessels, etc.) scar content.

A semiquantitative evaluation of newly formed vessels (20-fold magnification) and myofibroblasts, leukocytes and macrophages (40-fold magnification) within the scar was performed by immunohistochemistry with VWF (1:200), {alpha}-smooth muscle actin (1:50), CD45 (1:20) and CD68 (1:20) Dako antibodies (score 0=0, 1=1–50, 2=>50–100, 3=>100 vessels or cells per field); and collagen I/III ratio (two-fold magnification) by viewing Sirius red stained slides on polarized light microscopy, since type I collagen fibers appear orange or red, and the thinner type III fibers appear yellow or green [17] [score 1 (collagen III>I), 2 (collagen I=III), 3 (collagen I>III)]. All sections were subjected to antigen retrieval by heating in a microwave oven on high power for 8 min in 0.01 mol/l citrate buffer (pH 6.0) and then incubated, other than with the above-mentioned antibodies, with a mouse monoclonal anti-TGF-β1 -β2 and -β3 primary antibody to active TGF-β1 (150 µg/ml; 1:20, Genzyme Diagnostics, Cambridge, MA, USA). Immunoreactivity score was evaluated as percent of positive myocytes in the border area (defined as an area ≤5 mm far from the scar tissue) at 20-fold magnification (0=0%, 1=1–10%, 2=11–50, 3=>50% immunoreactive myocytes). For all immunohistochemistry studies, negative controls were performed by incubation of the sections with the omission of primary antibody and using the antibody diluent alone or the appropriate non-immune IgG in each case.

Histomorphometric analysis was performed also at the level of non-infarct, posterior LV wall in order to assess mean myocyte diameter (by counting 100 myocytes at a 20-fold magnification) and percent of interstitial fibrosis.

2.5 Statistical analysis
Data from all animals were expressed as mean±S.D. One-way analysis of variance (ANOVA) and Student t-test for unpaired data were used for differences between subgroups. Selected variables were compared by linear regression analysis. The difference was considered statistically significant at a level of p<0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions and clinical...
 References
 
Eight dogs died during ischaemia or reperfusion due to ventricular fibrillation. An anterior MI was obtained in 24 dogs (80%), with a transmural extension in 15 and subendocardial in 9; six dogs did not develop a MI: three (30%) in group I, two (20%) in group II and one (10%) in group III. Thus 24 plus five controls, for a total number of 29 hearts, were used for the final blind analysis of data. Main gross, histologic and immunohistochemistry data are reported in Table 1. Scar size ranged from 5 to 17% of the LV area (mean 11.5±4) and from 22 to 56% of the LV endocardial circumference (mean 37±9). A negative correlation was found between volume and M/V (p<0.0001, r = 0.76) and between expansion index and thinning ratio (p = 0.008, r = 0.47); and a positive correlation between posterior wall myocyte diameter and scar size in terms of % LV endocardial circumference (p = 0.003, r = 0.39).


View this table:
[in this window]
[in a new window]

  		Table 1 Main gross, histology and immunohistochemistry findings in the four groups

 
3.1 Efficacy of the experiment
The saline-treated MI hearts (group III) served to assess the efficacy of study protocol in achieving anterior MI. The MI was transmural in five and subendocardial in four, accounting for a mean scar size of 9.2±3.2% LV area and of 37.1±8.2% LV endocardial circumference. When compared to controls, group III showed a statistically significant lower thinning ratio (0.7 vs. 0.88, p = 0.001), higher expansion index (1.1 vs. 1, p = 0.05) and a higher myocardial cell diameter in the non-infarct posterior wall (16.4±1.3 vs. 14.5±1.4, p = 0.02) whereas no differences were found in terms of body weight, heart weight, volume, M/V, and posterior LV wall thickness. Histological and immunohistochemical study revealed a prevalence of type I collagen, a high score of myofibroblasts and newly formed vessels, and a low score of macrophages in the scar area, together with a high TGFβ score in the myocytes bordering the scar area.

3.2 Effect of drug on scar healing and LV remodeling
A homogenous distribution of MI was observed among the three groups. Group I comprised 7 MI out of 10 hearts (four transmural and three subendocardial); group II 8 MI out of 10 (six transmural and two subendocardial); and group III 9 MI out of 10 (five transmural and four subendocardial). No differences were found regarding scar size expressed either as %LV area (9.7±4.6 in group I vs. 11±5.2 in group II vs. 9.2±3.2 in group III, p = NS) or %LV endocardial circumference (38.1±10.2 in group I, 37.7±10.8 in group II and 37.1±8.2 in group III, p = NS). No difference between the three groups regarding scar size, transmural extent, expansion index, thinning ratio (Fig. 1) as well as histological (Fig. 2) and immunohistochemical variables (Fig. 3) were found.


Figure 1
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 Mean values of expansion index (a), thinning ratio (b) and percent fibrosis in the scar area (c). No significant differences among the three MI groups are visible. Group I, darusentan i.v. solus acute; Group II, darusentan i.v. solus acute+chronic oral; Group III, saline; Group IV, controls.

 

Figure 2
View larger version (70K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 Histological features at 6 weeks post-MI in group I (A, D), group II (B, E), and group III (C, F). (A, B, C) Transverse section of the heart showing healed anterior myocardial infarction stained in blue (Heidenhain trichrome x1); (D, E, F) polarized light microscopy showing a prevalence of orange–red collagen type I fibers (Sirius red x10).

 

Figure 3
View larger version (183K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Immunohistochemical features at 6 weeks post-MI in group I (A, D, G, L), group II (B, E, H, M), and group III (C, F, I, N). (A, B, C) Newly formed vessels in the scar (VWF x20); (D, E, F) active myofibroblasts ({alpha}-smooth muscle actin x40); (G, H, I) macrophages (CD68 x40); (L, M, N) TGFβ immunoreactivity of myocytes in the border area (TGFβ x20).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions and clinical...
 References
 
Early coronary recanalization after acute MI does not necessarily imply adequate myocardial reperfusion. This phenomenon, named "no-reflow", affects approximately 25% of patients and has both functional and clinical implications [18]. Thus, a therapeutic approach to acute MI should include measures to guarantee both patency and function of coronary microcirculation. We have recently shown that the ET antagonist darusentan, administered at reperfusion in a dog model of ischemia-reperfusion, exerts a beneficial effect on microcirculation, by reducing impairment of coronary flow and extent of "no-reflow" within the risk area, with a consequently relevant lower infarct size [8]. This beneficial effect is likely due to the reduction of various deleterious effects of ET during ischemia-reperfusion, i.e., potent vasoconstriction, neutrophil plugging, and increased permeability with interstitial edema. However, the positive effect of ET antagonists in the acute phase of MI seems to be at least in part counterbalanced by controversial effect on scar healing and LV remodeling. We performed this study to investigate the effect of immediate ET receptor antagonist administration in an experimental context simulating reperfusion obtained by primary PTCA in acute MI in man.

4.1 Experimental model and efficacy
Dogs subjected to 180 min LAD PTCA occlusion followed by reperfusion were sacrificed at 6 weeks post-MI. Before comparing our findings with those of previous experimental work on scar healing and LV remodeling, two major variables need to be emphasized, i.e., the animal model (dog vs. rat) and the method employed to induce MI (coronary artery permanent ligation vs. transient occlusion followed by reperfusion). First, rat infarcts produced by ligating the left main coronary artery involve the anterior LV free wall, are typically transmural, and comprise more than 35% of the LV free wall. Dog infarcts produced by LAD occlusion are predominantly subendocardial and much smaller due to the presence of collateral blood flow. The rate of infarct healing is also different in various species, i.e., healing is completed by 3 weeks in rats but continues beyond 6 weeks in dogs and humans. Regarding permanent occlusion vs. occlusion plus reperfusion, it is known that not only early coronary artery recanalization may salvage reversibly injured myocardium but also late recanalization may limit infarct expansion during the healing phase due to acceleration of infarct repair [19]. We used 3 h occlusion as this produces significantly more necrosis than shorter periods, with focal involvement of the subepicardial myocardium [20]. The validity of this approach was supported by achieving MI in 90% of hearts and a lower thinning ratio and a higher expansion index when compared to normal hearts.

According to Jugdutt and Ami [21] the healing process after MI in dogs with coronary artery ligation involves early cavity dilation and infarct expansion, followed later by infarct thinning and contraction but no significant compensatory hypertrophy at 6 weeks. Noteworthy, the values they obtained were very similar to ours both in terms of infarct size (11% vs. 9%) and thinning ratio (0.71 vs. 0.70), whereas the expansion index was significantly higher (1.32 vs. 1.1). Since major determinants of infarct expansion (i.e., transmural extension and infarct size) were comparable in the two series, according to the "open artery hypothesis" coronary reperfusion, instead of permanent ligation, could have positively affected the healing process in our model with better LV remodeling [19].

Regarding the post-MI healing process in terms of scar tissue composition, we found a prevalence of type I collagen, a high score of myofibroblasts and vessels, and a low score of macrophages, findings which are all in keeping with the timing of the healing process in dogs and humans at 6 weeks post-MI [17,22]. Moreover, cardiac myocytes bordering the infarct area showed a high immunoreactivity score for TGFβ. Increased expression of TGFβ1 mRNA and protein have been shown in myocardium bordering the infarct region early after MI suggesting a role in the cardiac wound healing response [23]. TGFβ1 is expressed in the heart and both cardiac myocytes and fibroblasts may release it and, similarly, TGFβ receptors are localized in both cardiac myocytes and non-myocytes [24]. TGFβ1 is a powerful initiator of the production of fibrillar collagens and other major extracellular matrix components in a variety of cell types, including cardiac fibroblasts which represent the predominant cell type in post-MI scar tissue thus mediating collagen deposition.

4.2 Effects of ET antagonists on scar healing
The data show that early treatment post-MI with the ETA selective receptor antagonist darusentan does not affect scar area histologic and immunohistochemical characteristics.

The potential benefits of drugs in congestive heart failure after MI have been evaluated in rats subjected to coronary artery ligation. Sakai et al. [9] demonstrated significant positive effects of late chronic infusion of ETA selective receptor antagonist BQ123 over 12 weeks, starting 10 days after coronary artery ligation, and documented a favorable effect on survival and cardiac function, by reducing LV end-diastolic pressure, right ventricular systolic pressure and central venous pressure, and preventing LV remodeling.

Mulder et al. [25] investigated the effect of chronic oral treatment with darusentan 10 or 30 mg/kg/day over 10 weeks, beginning 7 days after coronary artery ligation in rats. As compared to placebo, both doses improved LV and right ventricular function. Moreover, darusentan did not prevent cardiac hypertrophy but reduced collagen density in the non-infarcted subepicardial region of the LV. The same group [26] performed a trial with the non-selective ETA/B receptor antagonist bosentan, starting again 7 days after MI and they demonstrated reduced central venous pressure, LV end-diastolic pressure and systolic pressure, heart rate, as well as LV dilatation and collagen content at 2 months. At 9 months a higher survival rate than in controls was demonstrated, confirming results achieved with selective ETA receptor blockade [9].

On the other hand, studies initiating ET receptor blockade early after MI show a negative effect in the rat. Chronic oral treatment with EMD 94246 given for 8 weeks, starting on the day of MI resulted in an aggravation of LV remodeling in rats with large MI (>35%), as documented by a rightward shift of the passive pressure/volume curve (despite LV volume and mass being unchanged), but without effects on survival and LV or systemic hemodynamics [12]. Similar results were obtained when A-127222 was used as an antagonist [27]. Chronic treatment for 4 weeks with darusentan starting within 24 h after MI in rats resulted in negative effects on LV function which were ascribed to scar thinning, further LV dilatation and increase in LV end-diastolic pressure [11]. However, ETA receptor blockade led to significant improvement in right ventricular systolic pressure and right atrial pressure. Bosentan, when given early and continued for 8 weeks, resulted in attenuated LV dilatation and improved cardiac function in rats with large MI [10]. However, these effects were overall much less pronounced than in the studies in which treatment was initiated late after MI and no effects were demonstrated on LV cardiodynamics.

All these studies but one [10] provided evidence for a detrimental effect of early ET receptor blockade on cardiac function, due to excessive LV dilatation and unfavorable LV remodeling, effects that have been allegedly attributed to scar healing impairment as a consequence of the inhibition of collagen formation due to the known stimulating action of ET-1 on fibroblasts [28]. In particular, there is growing body of evidence that ET-1, via stimulating collagen type IV synthesis in fibroblasts, has an important role in extracellular matrix formation, and thus also in remodeling especially after injuries within the cardiovascular system [29]. It has been recently shown in human cardiac fibroblasts that ET-1, via the ETA receptor, stimulates collagen synthesis without having a mitogenic effect on the fibroblasts themselves, indicating an important role of ET-1 in the development of cardiac fibrosis and remodeling [30]. Noteworthy, none of the previous studies dealing with LV remodeling after MI specifically addressed scar area tissue composition, collagen content and TGFβ immunoreactivity. Moreover, as MI in the rat model is mostly huge (40–50% of the LV) and always transmural, the negative effect of early treatment needed to be investigated in another species such as the dog before conclusions could be drawn for humans. Furthermore, we chose a model of transient coronary occlusion with reperfusion due to its consistence with the clinical practice where coronary artery recanalization and myocardial reperfusion are achieved in the acute phase of MI by systemic thrombolysis or PTCA.

4.3 Limitations of the study
The experimental design does not allow us to determine whether the acute ETA receptor antagonist administration reduced final infarct size, as this was outside the scope of this study, which specifically aimed at investigating the post-MI scar healing process. Anyhow, we already showed in a comparable model the potential of darusentan to reduce both infarct size and no-reflow extent within the risk area [8]. Only a semiquantitative histology and immunohistochemistry analysis of TGFβ and collagen types I and III was performed in formalin-fixed myocardial samples whereas tissue expression of TGFβ isoforms as well as of collagen types I and II has not been assessed on mRNA or protein level by blotting techniques.


   5. Conclusions and clinical implications
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions and clinical...
References
 
The experiment clearly shows the absence of negative effects on scar healing after MI either in terms of histology or immunohistochemistry characteristics, indicating that the immediate chronic administration of ETA receptor antagonist in reperfused MI does not affect the scar healing process. These results bear even more importance in light of evidence of positive effects of ETA receptor antagonism after coronary artery recanalization and myocardial reperfusion in the acute phase of MI [8].

Time for primary review 20 days.


   Acknowledgements
 
The authors wish to thank Alessandra Dubrovich and Mauro Pagetta for the expert technical assistance and Chiara Carturan for the editorial assistance.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions and clinical...
 References
 

  1. Yanagisawa M., Kurihara H., Kimura S., et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature (1988) 332:411–415.[CrossRef][Medline]
  2. Tippins J.R., Antoniw J.W., Maseri A. Endothelin-1 is a potent constrictor in conductive and resistive coronary arteries. J. Cardiovasc Pharmacol. (1989) 13(Suppl. 5):S177–179.[Web of Science][Medline]
  3. Velasco C.E., Turner M., Inagami T., et al. Reperfusion enhances the local release of endothelin after regional myocardial ischemia. Am Heart J. (1994) 128:441–451.[CrossRef][Web of Science][Medline]
  4. Stewart D.J., George K., Costello K., et al. Increased plasma endothelin-1 in the early hours of acute myocardial infarction. J Am Coll Cardiol (1991) 18:38–43.[Abstract]
  5. Pernow J., Wang Q.-D. Endothelin in myocardial ischaemia and reperfusion. Cardiovasc Res (1997) 33:518–526.[Abstract/Free Full Text]
  6. Wang Q.D., Li X.S., Lundberg J.M., et al. Protective effect of non-peptide endothelin receptor antagonist bosentan on myocardial ischaemic and reperfusion injury in the pig. Cardiovasc. Res. (1995) 29:805–812.[Abstract/Free Full Text]
  7. Grover G.J., Dzwonczyk S., Parham C.S. The endothelin-1 receptor antagonist BQ-123 reduces infarct size in a canine model of coronary occlusion and reperfusion. Cardiovasc Res (1993) 27:1613–1618.[Abstract/Free Full Text]
  8. Galiuto L., DeMaria A.N., del Balzo U., et al. Ischemia-reperfusion injury at the microvascular level. Treatment by endothelin A-selective antagonist and evaluation by myocardial contrast echocardiography. Circulation (2000) 102:3111–3116.[Abstract/Free Full Text]
  9. Sakai S., Miyauchi T., Kobayasi M., et al. Inhibition of myocardial endothelin pathway improves long-term survival in heart failure. Nature (1996) 384:353–355.[CrossRef][Medline]
  10. Fraccarollo D., Hu K., Galuppo P., et al. Chronic endothelin receptor blockade attenuates progressive ventricular dilatation and improve cardiac function in rats with myocardial infarction: possible involvement of myocardial endothelin system in ventricular remodeling. Circulation (1997) 96:3963–3973.[Abstract/Free Full Text]
  11. Nguyen Q.T., Cernacek P., Stewart D.J., et al. Endothelin-A receptor blockade causes adverse left ventricular remodeling but improves pulmonary artery pressure postinfarction in the rat. Circulation (1998) 98:2323–2330.[Abstract/Free Full Text]
  12. Hu K., Gaudron P., Schmidt T.J., et al. Aggravation of left ventricular remodeling by a novel specific endothelin ETA antagonist EMD 94246 in rats with experimental myocardial infarction. J Cardiovasc Pharmacol (1998) 32:505–508.[CrossRef][Web of Science][Medline]
  13. Riechers H., Albrecht H.P., Amberg H.P., et al. Discovery and optimization of a novel class of orally active non peptidic endothelin-A receptor antagonists. J Med Chem (1996) 39:2123–2128.[CrossRef][Web of Science][Medline]
  14. Spieker L.E., Mitrovic V., Noll G., et al. Acute hemodynamic and neurohumoral effects of selective ETA receptor blockade in patients with congestive heart failure. J Am Coll Cardiol (2000) 35:1745–1752.[Abstract/Free Full Text]
  15. Munter K., Ehmke H., Kirchengast M. Maintenance of blood pressure in normotensive dogs by endothelin. Am J Physiol (1999) 276:H1022–H1027.[Web of Science][Medline]
  16. Eaton L.V., Bulkley B.H. Expansion of acute myocardial infarction: its relationship to infarct morphology in a canine model. Circ Res (1981) 49:80–88.[Free Full Text]
  17. Whittaker P., Boughner D.R., Kloner R.A. Analysis of healing after myocardial infarction using polarized light microscopy. Am J Pathol (1989) 134:879–893.[Abstract]
  18. Ito H., Maruyama A., Iwakura K., et al. Clinical implications of the "no-reflow" phenomenon: a predictor of complications and left ventricular remodeling in reperfused anterior wall myocardial infarction. Circulation (1996) 93:223–228.[Abstract/Free Full Text]
  19. Kim C.B., Braunwald E. Potential benefits of late reperfusion of infarcted myocardium: the open artery hypothesis. Circulation (1993) 88:2426–2436.[Free Full Text]
  20. Reimer K.A., Lowe J.E., Rasmussen M.M., et al. The wavefront phenomenon of ischemic cell death. I: myocardial infarct size vs. duration of coronary occlusion in dogs. Circulation (1977) 56:786–794.[Abstract/Free Full Text]
  21. Jugdutt B.I., Ami R.W.M. Healing after myocardial infarction in the dog: changes in infarct hydroxyproline and topography. J Am Coll Cardiol (1986) 7:91–102.[Abstract]
  22. Willems I.E.M.G., Havenith M.G., De Mey J.G.R., et al. The {alpha}-smooth muscle actin-positive cells in healing human myocardial scars. Am J Pathol (1994) 145:868–875.[Abstract]
  23. Thompson N.L., Bazoberry F., Speir E.H., et al. Transforming growth factor beta-1 in acute myocardial infarction in rats. Growth Factors (1988) 1:91–99.[Medline]
  24. Hao J., Ju H., Zhao S., et al. Elevation of expression of smads 2, 3 and 4, Decorin and TGFβ in the chronic phase of myocardial infarct scar healing. J Mol Cell Cardiol (1999) 31:667–678.[CrossRef][Web of Science][Medline]
  25. Mulder P., Richard V., Bouchart F., et al. Selective ETA receptor blockade prevents left ventricular remodeling and deterioration of cardiac function in experimental heat failure. Cardiovasc. Res. (1998) 39:600–608.[Abstract/Free Full Text]
  26. Mulder P., Richard V., Derumeaux G., et al. Role of endogenous endothelin in chronic heart failure: effect of a long-term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling. Circulation (1997) 96:1976–1982.[Abstract/Free Full Text]
  27. Pfeffer J.M., Finn P.V., Zornoff L.A.M., et al. Endothelin-A receptor antagonism during acute myocardial infarction in rats. Cardiovasc Drug Ther. (2000) 14:579–587.[CrossRef][Web of Science][Medline]
  28. Guarda E., Katwa L.C., Myers P.R., et al. Effects of endothelins on collagen turnover in cardiac fibroblasts. Cardiovasc Res (1993) 27:2130–2134.[Abstract/Free Full Text]
  29. Rizvi M.A.D., Katwa L.C., Spadone D.P., et al. The effects of endothelin-1 on collagen type I and III synthesis in cultured porcine artery vascular smooth muscle cells. J Mol Coll Cardiol (1996) 28:243–252.[CrossRef]
  30. Kirchengast M., Munter K. Endothelin-1 and endothelin receptor antagonists in cardiovascular remodeling. Proc Soc Exp Biol Med (1999) 221:312–325.[CrossRef][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Basso, C.
Right arrow Articles by Iliceto, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Basso, C.
Right arrow Articles by Iliceto, S.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?