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Eur J Cardiothorac Surg 2002;21:753-759
© 2002 Elsevier Science NL
a National Heart and Lung Institute Heart Science Centre, Harefield Hospital, Harefield, Middlesex UB9 6JH, UK
b Electron Microscopy Unit, Brompton Hospital, London, UK
c Cell Biology Department, Brompton Hospital, London, UK
Received 26 June 2001; received in revised form 8 January 2002; accepted 10 January 2002.
* Corresponding author. Tel.: +44-1895-828-550; fax: +44-1895-828-992
e-mail: mr.amrani{at}rbh.nthames.nhs.uk
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Abstract |
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 Top Abstract 1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Key Words: Endothelial function • Ischemia • Nitric oxide • Reperfusion • Vasoconstriction/dilation
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1. Introduction |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Acute administration of LA has been used as a strategy for protection of the endothelium during ischemia and reperfusion. We previously demonstrated that exogenous LA improved the postischemic recovery of cardiac mechanical function and coronary flow (CF) after cardioplegic arrest and ischemia by stimulation of NO production when given in the reperfusate [3,4]. In addition, other studies have shown improvement of recovery after ischemia when LA was administered during the period of ischemia/reperfusion [5–7].
On the other hand, chronic supplementation of LA is still controversial. Oral LA administration has been shown to play beneficial roles on attenuating hypertrophy [10], improving function in heart failure [11], reducing intimal hyperplasia [12], preserving isolated aortic ring relaxation and cardiac functions [13], although other investigations showed no beneficial effects on endothelium-dependent vasodilation and inflammation markers [14] and left ventricular function [15]. LA is contained in many foods, mainly in meat and fish. Therefore, if oral supplementation of LA effects on endothelial function after ischemia-reperfusion in healthy animals, more daily LA intake would be beneficial for healthy people to prevent cardiac events and to prepare cardiac surgery.
The aim of this study was to investigate the effects of chronic oral administration of LA on vascular recovery from ischemia-reperfusion injury following cold cardioplegic arrest in rats.
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2. Materials and methods |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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LA (4 mg/ml; Sigma Chemical Co., St. Louis, MO, USA) was administered in the drinking water in the LA group for 6 weeks until the experiments. The control rats had been kept for 6 weeks with normal drinking water. The amount of daily ingestion of water was recorded.
2.2. Experimental preparation and time course
The isolated rat heart preparation used in this study has already been described in detail elsewhere [16]. Briefly, Krebs–Henseleit bicarbonate buffer [3] was gassed with 95% oxygen and 5% carbon dioxide at 37°C and perfusion pressure was continuously maintained by keeping reservoirs 100 cm above the hearts through the experiment. Ischemic cardiac arrest was produced by clamping the aortic cannula. At this time, the hearts were subjected to a 10 ml hypothermic (4°C) coronary infusion with St. Thomas’ Hospital No. 1 solution and then kept immersed in the same solution for 4 h at 4°C maintained by cooling circuits. Time course is shown in Fig. 1
: vascular function study in Fig. 1a and morphological study in Fig. 1b. Blood samples were collected at the time of excision of the hearts. One blood sample (0.5 ml of whole blood) was mixed with 0.5 ml of 4% PCA and was frozen in liquid nitrogen followed by LA assay. Another sample was centrifuged and then plasma was isolated and stored at -70°C until NO assay.
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2.4. Vascular function studies
Endothelial and vascular smooth muscle functions were assessed through observations of preischemic and postischemic coronary flow responses to 5-HT (10-5 mol/l) and GTN (15 mg/l). After excision of the heart and aortic cannulation, Langendorff perfusion [16] was initiated for 20 min to allow steady coronary flow to be reached. The Langendorff infusion was switched to one containing an additional 10-5 mol/l 5-HT and then washed out with Krebs buffer. This was followed by perfusion with GTN (15 mg/l) and a washout period with Krebs buffer. Coronary flow was monitored proximally to the aortic cannula by an in-line electromagnetic flow probe (20-ml ECM2; Scalar, Delft, Holland), which was connected to its compatible flowmeter (MDL 1401; Scalar). After the ischemic period, the hearts were subjected to the same sequence of perfusion including the vascular function study. During perfusion with any substance, steady coronary flow was allowed to be reached before further perfusion was initiated. These time course is shown as protocol in Fig. 1a.
Coronary flow was expressed in milliliters per minute (ml/min). The vasodilatory responses to 5-HT and GTN were expressed as a percentage of change in the baseline coronary flow. Postischemic recovery of response to 5-HT and GTN was expressed as a percentage of individual preischemic control response. The postischemic recovery of coronary flow was expressed as the percentage of the preischemic value.
2.5. Vascular morphological studies
In a parallel series of experiments, endothelial and vascular smooth muscle morphology were assessed (Fig. 1b). After aortic cannulation, Langendorff perfusion was initiated at 37°C for 20 min. At the end of this period, the heart was subjected to cold ischemia similarly to vascular function studies. Then the heart was reperfused at 37°C for 20 min followed by perfusion-fixation with 2.5% glutaraldehyde in Krebs solution for 5 min. Next, the left ventricular free wall was dissected, and large thin slices (approximately 10x3x1 mm) cut transversely to include epicardial and endocardial surfaces were further fixed for 2 h in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). The tissue was postfixed and was embedded in Araldite CY212 as previously described [1]. Semithin (0.5–1 µm) sections were stained in 1% toluidine blue in a solution of 1% borax-50% ethanol, and examined with a light microscope. Four hearts were studied in each group and two separate blocks were examined. In another words, eight different sections were obtained in each group for morphological analysis.
To assess and compare the effects of the LA, vascular morphology was classified in a blind fashion as undamaged, mildly damaged, moderately damaged, or severely damaged as previously described [1]. Undamaged vessels had an intact endothelium lining the entire intimal surface with no microscopically visible damage. Smooth muscle was invariably well preserved in these vessels. Mildly damaged vessels were defined by the presence of an intact endothelium over 
90% of the luminal surface in which focal lifting of the endothelium or minor vacuolation was apparent. Smooth muscle was well preserved or showed only minor vacuolation. Moderately damaged vessels were defined by the loss of major portions (
50%) of the endothelium, and vacuolation and lifting were common. Underlying smooth muscle was frequently vacuolated. Severely damaged vessels were defined by the absence of endothelium over 50% of their surface with extensive structural damage of remaining cells. Smooth muscle was usually severely damaged in these vessels.
2.6. LA and nitrate/nitrite (NOx) levels
LA level was analyzed using a modified enzymatic method as previously described [17]. Blood samples were homogenized and microfuged followed by taking supernatant which was neutralized. After microfuging again, supernatant was obtained and was analyzed by a spectrophotometer using an enzymatic reaction: pyruvate+L-arginine+NADH
octopine+H2O+NAD. This reaction is catalyzed by octopine dehydrogenase. The change of absorbance at 340 nm was monitored and L-arginine level was calculated by comparing with the control curve. To determine total NO production in plasma, the amount of its breakdown product (nitrite) was assayed with a chemiluminescence method using an NO analyzer (Sievers 270, Colo) as previously described [3]. Nitrite is measured as an index of total NO production, as NO2- is the principal oxidation product in an aqueous solution devoid of any biologic contaminants. Release of LA and NOx was expressed in µmol/l.
2.7. Statistical analysis
Mann–Whitney U-test was applied for comparisons of vascular functions and levels of LA and NOx between the two groups. Paired t-test was used for the comparisons between the pre- and post-ischemic values. For the morphological analysis, two factor factorical analysis of variance was used. Values were given as mean±standard deviation (SD) and significance was assumed when P-value was less than 0.05.
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3. Results |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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In the control group, % response to 5-HT dropped from 63.7±17.4% at pre-ischemia to 13.8±19.9% at post-ischemia (P=0.0009). On the other hand, LA group preserved % response to 5-HT although it decreased from 62.5±49.0% at pre-ischemia to 39.0±26.2% at post-ischemia (P=0.13; NS). In addition, recovery of percentage response to 5-HT (post-/pre-ischemic value) was significantly greater in the LA group (97.0±65.6%) than in controls (21.5±25.7%, P=0.015).
Similarly, % response to GTN dropped from 56.2±17.9% at pre-ischemia to 27.9±14.7% at post-ischemia in the control group (P=0.0004). However, in the LA group, % response to GTN was not significantly altered: 61.5±32.3% at pre-ischemia and 53.5±28.6% at post-ischemia (P=0.50; NS). In addition, recovery percentage response to GTN (post-/pre-ischemic value) was significantly higher in the LA group (124.5±117.6%) than in control (47.7±16.6%, P=0.021). These results are compared graphically in Figs. 3–5 .
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4. Discussion |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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Others and we have shown that LA, the physiologic substrate for NO production, enhances the postischemic level of NO and coronary endothelium-dependent dilatation, suggesting that NO production and/or release is reduced following ischemia and reperfusion [3–6]. It is also well known that LA enhances release of growth hormone, glucagon and insulin, resulting in an improved glucose metabolism [18]. In healthy blood vessels, NO is produced mostly in the endothelial cells, by NO synthase(s) (NOS), which are expressed constitutively (basal level of NO). The induced NOS causes the production of larger amounts of NO (stimulated level of NO) from LA than that produced by constitutive NOS from endothelial cells [19]. The endothelium, particularly endothelium-derived NO, not only modulates the tone of the underlying vascular smooth muscle but also inhibits several proatherogenic processes, including smooth muscle proliferation and migration, platelet aggregation, oxidation of low density lipoproteins (LDL), monocyte and platelet adhesion, and synthesis of inflammatory cytokines [9].
Regarding the acute effects of LA, we previously demonstrated that exogenous administration of LA improved the post-ischemic recovery of cardiac mechanical function and CF after cardioplegic arrest and ischemia by stimulation of NO production when given in the reperfusate [3,4]. Other studies also have shown improvement of recovery after ischemia when LA was administered during the period of ischemia/reperfusion [5–7]. Administration of LA reduced the ischemia-reperfusion injury [5] and infarction size [6,7], and ameliorated the endothelial dysfunction caused by hypercholesterolemia [20], atherosclerosis [21], and aging [22]. As for subacute effects of LA supplementation, Mitani et al. [23] reported that daily intraperitoneal injection of LA ameliorated chronic pulmonary hypertension and pulmonary vascular remodeling in rats, possibly by modifying the endogenous NO production. In spite of these beneficial cardiovascular effects of LA, the mechanism of LA-NO protection pathway has not been clearly defined. One possible explanation may be that NO synthesis is inhibited, especially under pathologic conditions, by LA analogues (such as asymmetric dimethyl-LA), which compete with endogenous LA: it was suggested that the high dose of exogenous LA (both oral and intravenous) may overcome this effect [24].
Not only acute/subacute effects but also chronic effects of LA administration have been reported. Matsuoka et al. [10] revealed that chronic LA supplementation attenuated cardiac hypertrophy in spontaneously hypertensive rats independently of blood pressure and increased myocardial content of cGMP and nitrate/nitrite. Rector et al. [11] showed that supplemental oral LA for 6 weeks in patients with heart failure increased the forearm blood flow and the distances during a 6-min walk test. Furthermore, LA also improved arterial compliance and reduced circulating levels of endothelin. Chen et al. [12] reported that oral LA for 2 weeks reduced intimal hyperplasia in balloon-injured rat carotid arteries: This inhibitory effect of LA may be mediated by increased production of NO which inhibits platelet aggregation, leukocyte adhesion, and smooth muscle cell growth, thereby reducing intimal hyperplasia formation. In contrast, Blum et al. [14] showed that oral LA in healthy postmenopausal women (9 g/day for a month) did not change the serum NO level, inflammation markers, and brachial artery endothelium-dependent dilator responses to hyperemia. In addition, Bartunek et al. [15] revealed that oral LA supplementation depressed in vivo LV systolic function in LVH rats and markedly blunted the contractile response to beta-adrenergic stimulation by long-term stimulation of NO-cyclic GMP signaling.
Interestingly, the present study showed that LA preserved not only endothelium-dependent but also endothelium-independent function: in the intact endothelium, 5-HT causes endothelium-dependent vasodilatation through the release of NO, on the other hand, GTN is an endothelium-independent vasodilator [1]. Though the exact mechanism is unclear, we speculate that the probable cause was the LA-NO pathway in the endothelium, which might indirectly protect the vascular smooth muscle by increased NO: stimulated NO level could be increased in the LA group during/after ischemia despite basal (pre-ischemic) NO level was not.
In spite of better results of vascular function in LA group, there were no detectable differences in the vascular morphology. Possible explanation is that ischemia-reperfusion induces functional vascular injury before morphological damage becomes apparent. Function study may be more sensitive than morphological approaches for detection of the early stage of injury. If the degree of ischemia was more severe, then morphological differences between the two groups might emerge. As the degree of recovery of reduction in coronary flow, and of basal and stimulated release of NO, diminished with age [8], further study using animals of different age may be needed to reveal the effects of LA.
The present study has several limitations. Our observation was made in healthy animals. Other studies are needed to investigate the effects of LA in atherosclerotic, hypercholesterolemic or failed hearts. Our results showed that ischemia-reperfusion injured endothelium and vascular smooth muscle and this impairment was improved by chronic oral administration of LA. Chronic administration would mimick condition for oral chronic intake by healthy people, whilst acute administration, which is usually given in the cardioplegia or reperfusate, would mimick condition for cardiac surgery as previously shown [3,4] or acute cardiac events. In addition, chronic administration could have a prophylactic effect on the onset of cardiac events as well as in cardiac surgery by having better endothelial function after ischemia-reperfusion. This suggests that healthy individuals could benefit from prophylactic chronic intake of LA. Consequently, there are great advantages of chronic LA administration against acute one, because it is easy to increase LA intake from foods (mainly meat and fish) and target people are massive. In the present study, we did not compare the acute and chronic administration of LA because we considered that the potential targets were different between them: treatment for cardiac patients and preventive medicine for healthy individuals, respectively. However, the comparison of acute and chronic administration may be needed to investigate the difference of efficacy between them. As how much and how long should be appropriate for chronic LA administration was not investigated, those matters would be future works. In our study, crystalloid solution was used as perfusion, therefore, effects of platelet adhesion and neutrophils were ruled out. Another limitation relates to the fact that other time points of vascular function test could be interesting, e.g. 1 h later. However, the hearts would have been used for approximately 6 h, and the cardiac edema would be markedly enhanced.
In conclusion, the postischemic recovery of endothelial and vascular smooth muscle functions was significantly ameliorated by chronic oral administration of LA. Stimulated NO release in response to 5-HT could reflect the better endothelial and smooth muscle protection of LA group. Therefore, the vascular function was well preserved by stimulated NO release via LA-NO pathway during ischemia-repurfusion.
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References |
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TopAbstract1. Introduction2. Materials and methods3. Results4. DiscussionReferences |
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