Atherosclerosis 155 (2001) 53 – 59 www.elsevier.com/locate/atherosclerosis
MK-954 (losartan potassium) exerts endothelial protective effects against reperfusion injury: evidence of an e-NOS mRNA overexpression after global ischemia Antonio Barsotti a, Pericle Di Napoli a, Alfonso A. Taccardi a, Rita Spina a, Liborio Stuppia b,c, Giandomenico Palka b, Renato C. Barbacane d, Raffaele De Caterina a, Pio Conti d,* a
Laboratory of Experimental Cardiology, Department Clinical Sciences and Bioimaging, Uni6ersity of Chieti, Chieti, Italy b Department Biomedical Sciences, Medical Genetic Uni6ersity of Chieti, Chieti, Italy c Institute Cytomorphology, CNR Uni6ersity of Chieti, Chieti, Italy d Immunology Di6ision, Department of Oncology and Neurosciences, Uni6ersity of Chieti, Via dei Vestini, 66013 Chieti, Italy Received 30 November 1999; received in revised form 25 April 2000; accepted 28 April 2000
Abstract Background: the cardiac Renin–Angiotensin system (RAS) plays an important role in the regulation of coronary flow and cardiac function and structure in normal and pathological conditions such as ischemia– reperfusion (I/R) injury. The aim of this study was to investigate the effects of the Angiotensin II type 1 (AT-1) receptor antagonist MK-954 (losartan potassium) on postischemic endothelial dysfunction and NOS mRNA expression (inducible nitric oxide synthase, iNOS; endothelial nitric oxide synthase, eNOS) in isolated working rat hearts. Methods: isolated working rat hearts were subjected to 15 min global ischemia and 180 min reperfusion. MK-954 was added to perfusion buffer (a modified Krebs– Henseleit solution) at 1 mM concentration. We assessed functional parameters, creatin kinase (CK) release, heart weight changes, microvascular postischemic hyperpermeability (FITC-albumin extravasation) and morphological ultrastructural alterations. eNOS and iNOS mRNA levels were also detected by the means of multiplex RT-PCR technique using glyceraldehyde-3-phosphate dehydrogenase (G3PDH) gene as internal control; results were expressed as densitometric ratio. Results: in Losartan-treated hearts we observed a significant reduction of postischemic contractile dysfunction, CK release and myocardial ultrastructural damage; postischemic FITC-albumin extravasation was significantly reduced respect to controls. Moreover, 1 mM Losartan produced a significant reduction of eNOS/G3PDH respect to untreated hearts submitted to I/R. Regarding iNOS/G3PDH ratio, no significant changes were detected in Losartantreated hearts compared with controls. Conclusions: our study revealed that Losartan treatment before ischemia, and during reperfusion, is able to reduce the reperfusion injury of the rat heart by reducing mechanical and microcirculatory dysfunction and necrotic cell death, ameliorating cardiac ultrastructure and endothelial protection, probably inducing eNOS over-expression and reducing post-ischemic hyperpermeability of coronary microcirculation. © 2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Losartan; Endothelium; Nitric oxide; Reperfusion damage
1. Introduction The cardiac Renin – Angiotensin system (RAS) plays an important role in the regulation of coronary flow and cardiac function and structure in normal and pathological conditions [1]. Angiotensin II (Ang II) * Corresponding author. Tel.: +39-0871-3555293; fax: + 39-871561635. E-mail address:
[email protected] (P. Conti).
elicits several physiological effects that could exacerbate ischemia –reperfusion (I/R) injury [2]. Ang II interacts with two pharmacologically distinct subtypes of cellsurface receptors, Ang II type 1(AT-1) and type 2 (AT-2) [3,4]. Both AT-1 and AT-2 receptors are expressed in the heart and modulate I/R injury [5,6]. Although ACE-inhibitors are beneficial during myocardial ischemia, their effects on reperfused myocardium remain controversial [7]. The alternative strategy to reduce the negative effects of endogenous Ang II stimu-
0021-9150/01/$ - see front matter © 2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 0 ) 0 0 5 3 3 - 5
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lation is the use of selective Ang II type 1 (AT-1) and type 2 (AT-2) receptor antagonists. MK-954 (Losartan potassium) is the first of a new class of non-peptide Ang II AT-1 specific receptor antagonists. Its beneficial properties in chronic heart failure and ischemic disease have been previously reported [8]. Conflicting data exist regarding its role in I/R damage [9]. Yang et al. [6] reports that reperfusion injury induces an increase in the number of Ang II receptors, specifically the AT-1 subtype. The increased AT-1 receptor expression might relate to tissue repair or to fibrogenic response to tissue injury after ischemia. The effect of acute Losartan treatment on nitric oxide synthesis and endothelial dysfunction that characterized the ischemic-reperfused myocardium is still unknown. The aim of this study was to investigate the effects of the Ang II AT-1 receptor antagonist MK-954 on postischemic endothelial dysfunction and NOS mRNA expression (inducible nitric oxide synthase, iNOS; endothelial nitric oxide synthase, eNOS) in isolated working rat hearts.
2. Materials and methods
2.1. Animals and perfusion technique Adult male Wistar rats (n =45) were anesthetized with a mixture of ether and air. After injection of 1000 U of heparin in the femoral vein, hearts were quickly excised, weighed and perfused with working heart technique [10]. Modified Krebs – Henseleit solution (KH: 108 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2, 11 mM glucose, 287 mOsm) was used as perfusion medium. The buffer was bubbled in 95% O2 and 5% CO2, maintained at 37°C and at pH 7.4. Preload (height of atrial chamber) and afterload (height of aortic chamber) were set at 20 and 72 cm H2O, respectively. Aortic and coronary flows (ml/min) were measured, collecting aortic chamber overflow and heart chamber effluent into graduated cylinders. Aortic pressure (mmHg) was monitored through a membrane transducer (TNF-R, Viggo-Spectramed, Oxnard CA) connected to a side arm of the aortic cannula. Heart rate (bpm) and rhythm were determined with an epicardial ECG (Cardioline 350/1, Milan, I). Minute work (mmHg × ml/min) was computed as the product of cardiac output (sum of aortic and coronary flows) and peak aortic systolic pressure.
2.2. Experimental protocol: Rat hearts were divided into three groups (n: 10 per group, Fig. 1). Group A, control hearts subjected to 225 min perfusion in working heart mode; Group B,
hearts subjected, after stabilization (20 min), to 15 min of global ischemia, 10 min Langendorff reperfusion and 180 min of reperfusion in working heart; Group C, hearts subjected, after stabilization, to 15-min of global ischemia, 10 min Langendorff reperfusion and to 180 min of reperfusion with Losartan 1 mM (MSD, Italy) added to perfusion buffer at the beginning of experimental procedures. The Losartan concentration used in this study has previously been shown to occupy virtually 100% of the AT-1 receptors [11].
2.3. Heart weight changes All the hearts were weighed before and reweighed after experimental procedures. Myocardial reperfusion edema was estimated through percent heart weight gain (heart weight before/heart weight after I/R × 100).
2.4. Enzyme release e6aluation Myocardial necrosis enzyme assay of coronary effluent was performed during stabilization (20 min), Langendorff reperfusion (45 min), and working heart reperfusion (55, 65, 75, 85, 95, 105, 145, 185 and 225 min). Creatinekinase (CK) activities from eluate samples were determined using commercial kits (Boehringer Mannheim, Milan, I). Data were reported as IU/ml/g wet weight.
2.5. Endothelial permeability Additional rat hearts of groups B and C (n= 5) were subjected, after ischemia, to 20 min Langendorff reperfusion with 75 mg fluorescein isothiocyanate-albumin (FITC-albumin, Sigma, Milan, I) dissolved in 200 ml KH to assess the microvascular permeability changes. Hearts were then reperfused with standard KH in Langendorff for 2 min, in order to eliminate intravascular fluorescence, and reweighed. In group A, the same protocol (n= 5) was performed starting at 35 min. Microvascular permeability changes were determined on FITC-albumin hearts by means of fluorescent microscopy. Ventricles were cut transversely into four to five blocks. Tissue blocks were immediately embedded in medium (O.C.T. Compound, Miles, Elkhart, IN) and stored at − 80°C. Tissue blocks were mounted on a specimen holder in a Slee microtome-cryostat maintained at − 35°C and oriented so that capillaries and muscle fibers could be cross-sectioned. Ten 5-mm sections were obtained from each tissue block and placed on a labeled slide, which was pre-warmed on a hot plate. The slides were immediately returned to the hot plate for drying, placed in a dark box for at least 1 h and then viewed and photographed at 40× under fluorescent light. FITC-albumin extravasation was quantified per section using an image analysis system
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(Image-Pro plus, Media Cybernetics, Silver Spring, MD) and expressed as integrated optical intensity (IOI) units according to Ramirez et al. [12].
2.6. Ultrastructural e6aluation Myocardial specimens (n =3 per group) were fixed in 2.5% glutaraldehyde for at least 3 h. Specimens were post-fixed in 1.33% osmic acid, dehydrated in graded series of ethanols and embedded in Epon 812 Resin. Subsequently, thin sections were taken (30 – 40 sections per specimen) stained with uranyl acetate and lead citrate, and studied by electron microscopy. An average of 30 fields per section were examined and photographed at 4.500× magnification. Histological changes (interstitial space area, mitochondria damage score) were analyzed using a computerized image analysis system (Image-Pro plus, Media Cybernetics, Silver Spring, MD). To quantify mitochondrial damage, a mitochondrial score index was used [13]. Interstitial space changes were quantified as the area of interstitial space relative to total myocardial area×100; these data were expressed as percent changes with respect to normal heart specimens (n = 5), serving as control.
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2.7. RNA extraction and RT-PCR analysis Multiplex RT-PCR was used to determine eNOS or iNOS mRNA levels in rat ventricular tissue. Rat myocardial samples (n=5 per group), fixed in liquid nitrogen and stored at − 80°C, were homogenized in 800 ml of RNA FAST solution (Celbio, Italia) as previously reported [14,15]. Total RNA was isolated as outlined by the manufacturer. RNA was dissolved in DEPC treated water and quantified spectrophotometrically at 260 nm. First-strand cDNA was generated by adding RNA (0.1 mg) to a mixture containing 1 mM dNTP, 1U/ml RNase inhibitor, 2.5 U/ml Moloney murine leukemia virus reverse transcriptase, 2.5 mM Random Examers, 5 mM MgCl2, 10× PCR buffer II in a final volume of 20 ml. Reverse transcription was carried out at 42°C for 1 h followed by heat inactivation of the reverse transcriptase at 99°C for 5 min. For semi-quantitative analysis of iNOS and eNOS mRNAs, rat G3PDH gene served as an internal control in calculation of densitometric results. The G3PDH and eNOS (iNOS) mRNAs were co-amplified by multiplex PCR. The PCR solution contained 10 ml of first-strand cDNA, 4 ml 10× PCR buffer, 2 mM MgCl2, 0.15 mM
Fig. 1. Experimental protocol. Stab., stabilization in working heart mode; W.H., working heart perfusion; Lang., Langendorff perfusion; FITC-Alb, Langendorff perfusion with FITC-albumin; Isch., global normothermic ischemia.
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Fig. 2. Haemodynamic parameters. (A) systolic aortic pressure (SAP); (B) coronary flow (CF); (C) minute work (MW); (D) coronary resistance (CR). Data are reported as mean +S.D.
sense (5%-ACC ACA GTC CAT GCC ATC AC-3%) and antisense (5%-TCC ACC ACC CTG TTG CTG TA-3%) G3PDH primer, 0.15 mM sense (5%-CGA GAT ATC TTC AGT CCC AAG C-3%) and antisense (5%-GTG GAT TTG CTG CTC TCT AGG-3%) eNOS or iNOS (sense: 5%-T, …, T GTG CCT TTG CTG ATG AC-3% and antisense: 5%-CAT GGT GAA CAC GTT CTT GG-3%) primers, 2U Taq DNA polymerase (Celbio) and water to a final volume of 50 ml. These samples were overlayed with mineral oil and subjected to 35 cycles of 95°C for 60 s, 60°C for 60 s, and to one cycle of 72°C for 7 min. PCR products were run on 2% agarose gel electrophoresis and visualized by ethidium bromide staining and exposure to ultraviolet light. The band density (G3PDH, eNOS and iNOS) was analyzed using a computerized densitometric system (Bio Rad Gel Doc 1000, Milan, I); the ratio of the eNOS and iNOS to G3PDH was determined.
2.8. Statistical analysis Student’s t-test was used for two-group comparisons (heart weight, ultrastructural data, vascular permeability, changes in gene expressions) and analysis of variance (ANOVA) for multiple-group comparisons (haemodynamic parameters, CK release), after the as-
sessment of normality of distribution. The probability of null hypothesis B 5% (P B 0.05) was considered statistically significant. All the results are reported as mean9 S.D.
3. Results Losartan treatment produced a significant reduction of postischemic mechanical dysfunction (Fig. 2); in particular, coronary flow was significantly higher in group C, compared to group B. Additionally, in group C, a significant decrease of coronary resistance (Fig. 2) was observed associated with a minor decrease in heart weight (A, 89 2%; B, 369 9%; C, 149 5.4%, PB0.001 vs. B). In group C, a reduction of CK release (Fig. 3), index of necrotic cell death, compared with group B, was also detected in coronary effluent. Postischemic microvascular permeability changes are reported in Fig. 3. A significant reduction of FITC-albumin extravasation was detected (P B 0.01 vs. B) in 1 mM Losartan treated hearts (group C). Electron microscopy analysis revealed (Fig. 4) in group B a significant increase (P B 0.001 vs. C) in the interstitial area (interstitial edema), especially in the perivascular area. Morphological evaluation of group B
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also demonstrated signs of ischemic damage in terms of areas of necrosis with disruption of normal interfibril geometry, swollen cells and mitochondria, with severe alterations of the cristae. Endothelial damage was also evident in terms of swelling and membrane disruption. The myocytic and endothelial damage was decreased in group C. eNOS and iNOS mRNA expression results are shown in Fig. 5. In rat hearts, ischemia induces a reduction of eNOS mRNA levels and significantly increases the G3PDH/eNOS ratio (P B 0.05 vs. A). Losartan administration determines an increase of eNOS mRNA levels and a significant decrease (PB 0.05) of G3PDH/eNOS ratio compared with group B. Regarding iNOS mRNA expression no significant changes were detected in both the groups.
Fig. 4. The effects of Losartan on ultrastructural mitochondrial damage score index and interstitial space area. Data are expressed as mean +S.D.
4. Discussion
Fig. 3. Top: release of creatine kinase (CK) in coronary effluent (IU/ml/min); Bottom, evaluation of permeability changes (FITC-albumin diffusion) of coronary microcirculation (IOI).
In isolated working rat hearts, our study revealed that Losartan, an in vivo active molecule with active metabolites [16], improves coronary flow, mechanical left ventricular performance and myocardial recovery after global normothermic ischemia. These cardioprotective effects appear to be associated with a direct endothelial protection in terms of postischemic hyperpermeability reduction of the coronary microcirculation, endothelial cell ultrastructural damage and modulation of NOS expression. These results, performed in a blood-free perfusion buffer and in an isolated heart model, emphasize the role of local or paracrine RAS in the evolution of ischemia-reperfusion damage. Ang II is a potent coronary artery constrictor in the rat that contributes to the ischemic coronary
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events through its hemodynamic [17,18] and endothelial [19] effects. In addition, earlier studies demonstrated an increase of myocardial AT-1 receptor expression immediately after I/R in isolated working rat hearts [5]. The marked increase in AT-1 receptor expression after I/R most likely contributes to the increase in coronary vascular resistance and cardiac dysfunction after I/R. Losartan is a highly specific Ang II AT-1 antagonist and the cardioprotective effects of this drug may be explained by the fact that it prevents the untoward effects of Ang II on ischemic reperfused hearts. Our data are in agreement with Paz et al. [7]; the authors reported that Losartan significantly attenuated the I/Rinduced changes in coronary flow and cardiac function in isolated rat hearts. Regarding the possible mechanisms of the anti-ischemic effect of Losartan, we evidenced a significant endothelial protection and an over-expression of eNOS mRNA (decrease of G3PDH/ eNOS ratio) in the Losartan-treated hearts; in contrast, Losartan has not induced changes in iNOS mRNA expression. NO is an important paracrine substance that has a significant role in the modulation of I/R
injury, endothelial function and coronary microcirculation [20 –24]. Due to these multiple actions, it has been hypothesized that decreased NO is implicated in myocardial damage induced by I/R [25 –27], and that at least part of the cardioprotective effects of Losartan might be mediated by NO. On theoretical grounds, it is likely that eNOS upregulation by Losartan has a positive effect after I/R because of the vast endothelial protective effects of NO. The over-expression of eNOS gene may induce an increase of the endothelial NO synthesis, responsible, at least in part, for the observed improvement of post-ischemic coronary flow and endothelial function (microvascular permeability). On the other hand, the inhibition of postischemic iNOS over-expression exerts positive effects in ischemic conditions. The relationship between NO and local RAS is complex and the mechanisms leading to the production of NO by Ang II are controversial [28 –30]; Ang II stimulates inositol phosphate production and increases intracellular calcium concentration [31]. This increase in intracellular calcium induced by Ang II might be re-
Fig. 5. Constitutive eNOS, iNOS, and G3PDH gene expression in ventricular tissue in various experimental settings. (A) control heart in non-ischemic conditions; (B) heart subjected to I/R; (C) heart subjected to I/R treated with Losartan 1 mM. mRNA steady state levels are calculated as the densitometric ratio of eNOS and iNOS to G3PDH serving as internal control. Data are expressed as mean 9 S.D. Top, iNOS and eNOS mRNA expression; values are expressed as iNOS/G3PDH and eNOS/G3PDH densitometric ratio (n =5). *PB 0.05 versus C. Bottom, representative RT-PCR analysis of eNOS, iNOS and G3PDH mRNA.
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sponsible for the activation of iNOS that characterizes the reperfusion period and exerts deleterious effects by increasing peroxidative damage of cell membranes and apoptotic cell death [32]. In conclusion, our study reveals that Losartan treatment before ischemia, and during reperfusion, is able to reduce the reperfusion injury of the rat heart. The main effects are represented in (i) reduction of mechanical and microcirculatory dysfunction, (ii) amelioration of cardiac ultrastructure and a reduction of necrotic cell death; (iii) endothelial protection, probably due to the eNOS over-expression and (iv) reduction of the post-ischemic hyperpermeability of coronary microcirculation, that characterizes early reperfusion and exerts an important pathophysiological role in the no-reflow phenomenon.
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