1 Divison of Health Prevention Science, College of Public Health and
2 Cardiovascular and Thoracic Surgery and The Sarver Heart Center, School of Medicine, University of Arizona, Tucson, AZ 85724, USA
Received 17 January 2002;
in revised form 10 May 2002; in revised form 5 June 2002;
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ABSTRACT |
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INTRODUCTION |
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Alcohol misuse has a negative impact on human health. However, the relation of alcohol intake to mortality resembles a J-shaped curve. The higher the intake, the higher the mortality, with the exception that abstainers have higher mortality than moderate drinkers (Poikolainen et al., 1996). Epidemiological studies reported that moderate consumption of ethanol reduces the risk of coronary heart disease, sudden cardiac death, and ischaemic stroke (Stampfer et al., 1988
; Iso et al., 1995
; Poikolainen et al., 1996
; McKee and Britton, 1998
; Van Tol and Hendriks, 2001
). It is seen that regular moderate drinking tends to have a beneficial effect on the heart. The mechanisms for cardiovascular protective effects are not well established. We propose that moderate ethanol consumption could modulate coronary microvascular function.
Fourteen per cent of HIV-infected patients misuse alcohol (Welch, 2000). Moderate ethanol intake may modulate cardiovascular alterations in AIDS. In this study, we therefore investigated the combined effects of AIDS and ethanol consumption on coronary microvascular integrity.
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MATERIALS AND METHODS |
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Heart isolation
The animal model used to directly visualize coronary microcirculation is a modified Langendroff heart preparation (McDonagh, 1983). The procedure is as follows: After 3 months of intervention, mice were anaesthetized with sodium pentobarbital (55 mg/kg i.p.). The abdomen was opened to expose the abdominal aorta. Immediately, a PE 10 catheter was inserted and advanced to the aortic arch. The vena cava was then cut and cold cardioplegic solution (Abbotts Cardioplegic Solution for Cardiac Perfusion) was injected immediately through the catheter to arrest and protect the heart. After the heart stopped beating, a medial sternotomy was rapidly performed to expose the heart. Loose ligatures were placed around the right innominate artery and ascending aorta. Heparin (15 U) was injected into the right atrium. The ligatures around the subclavian and common carotid arteries were tied, and a PE 50 catheter was inserted into the innominate artery. The catheter was advanced toward the heart, until the tip extended just into the aorta. The catheter was secured and connected to the extracorporeal perfusion system. A small hole was cut in the right atrium. The aortic ligature was tied quickly, ensuring that all perfusate flow was retrograde to the coronary circulation. Then, the heart was removed from the thoracic cavity and placed on a heated Lucite stage for intravital fluorescence microscopy of the left ventricular epicardial microcirculation. The isolated hearts were perfused with a physiological solution that maintained normal cardiac and normal coronary microvascular functions.
Preparation of diluted whole blood for perfusate
Donor rats (450500 g) were anaesthetized with ether, and 6 ml of arterial blood were withdrawn immediately into a heparinized syringe via cardiac puncture. The rat blood was then diluted 1:1 with Krebs-bicarbonate solution. A small aliquot of diluted whole blood was used to measure pH, PO2, PCO2, haematocrit (Hct), leucocyte and platelet counts. Typical values obtained from the diluted whole blood were: pH 7.377.45, PO2 100125 mmHg, PCO2 3040 mmHg, Hct 21%, leucocyte counts 5.2 x 103/µl, platelet counts 2.0 x 105/µl.
Preparation of fluorescein isothiocyanate (FITC)BSA
The preparation was described previously (McDonagh and Williams, 1984). The FITC was first conjugated to albumin as follows: 1.25 g of albumin were dissolved in 18.75 ml of carbonate-bicarbonate buffer (CBB, pH 9.0). In a glass beaker, 0.0625 g of FITC were added to 6.25 ml of CBB and stirred until the FITC was completely dissolved. The albumin/CBB and the FITC/CBB solution were then mixed and covered with foil. The mixture was stirred at a slow speed overnight at 4°C. The following day, 25 ml of FITCBSA was run down a Sephadex column (Sephadex G-25, medium, Amersham Pharmacia Biotech; 100 ml Cap. Aldrich Flash-Chromatography Columns) to separate the conjugated from unconjugated FITC. Fifteen millilitres of conjugated FITC were collected and concentrated with an Amicon Centriprep (molecular weight cut-offs 10 kDa). The final conjugated FITCBSA solution was poured into a sterile tube, bringing the total volume up to 10 ml with sterile phosphate-buffered saline.
Measurement of coronary microvascular permeability to macromolecules
The isolated perfused mouse heart was oriented on the microscope stage with the free wall of the left ventricle facing up. A large coronary vein that courses along the left ventricle from apex to base was used to orient the heart on the stage. The aortic catheter was then connected to the syringe pump containing the perfusate. The coronary perfusate consisted of a mixture of 47.5% fresh whole rat blood obtained by cardiac puncture from a donor rat, 47.5% Krebs-BSA and 5% FITC BSA. The [K+] of the final perfusate solution was 30 mM, to ensure cardiac arrest while observing the coronary microcirculation. The perfusate blood gas was measured before perfusion. During constant flow perfusion at a flow rate of 0.3 ml/min, the coronary perfusion pressure was monitored (Pressure Monitor BP-1) via a sideline.
Five coronary microvascular fields were observed after 5, 15 and 25 min of perfusion using intravital fluorescence microscopy (Ritter and McDonagh, 1997). Venular fields were brought into clear focus and videotaped at each time point. After 30 min of observation, the perfusion was stopped. The videotaped results were analysed using Dazzle DVC and Adobe software (Adobe Photoshop 5.5). Five to six fields/heart/ time points were determined for the O/I ratio. The O/I ratio was used to quantify transcoronary FITCBSA leakage (Fig. 1
).
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RESULTS |
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DISCUSSION |
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Epidemiological studies demonstrate a significant protective effect of moderate alcohol consumption on the incidence of cardiovascular diseases. Possible mechanisms to explain the cardioprotective effect of ethanol are vascular relaxation, HDL elevation, lowering fibrinogen level, modulating platelet function, and anti-thrombotic properties (Redmond et al., 2000; Van Tol and Hendriks, 2001
). Recently, Arbabi et al.(1999)
found that ethanol inhibited proinflammatory cytokine, TNF and IL-8, secretion. A lower level of proinflammatory cytokines reduces their stimulating effect on neutrophils and platelets. Preventing chronic neutrophil and platelet activation benefits the coronary microcirculation. Ethanol also suppresses cytokine-induced iNOS expression (Syapin et al., 2001
), which may involve a mechanism of cardiovascular endothelial cell apoptosis. In addition, ethanol is a vaso-relaxant (Fitzpatrick et al., 1993
). This effect may be related to ethanol increasing both vascular endothelial growth factor mRNA expression and protein expression (Gu et al., 2001
). Overall, the cardiac protective effect of moderate ethanol consumption may be mediated by cytokine modulation and vaso-relaxation. In our study, chronic ethanol consumption alone did not alter coronary permeability to macromolecules. When murine AIDS mice were exposed to ethanol, coronary resistance was consistently maintained over the entire perfusion period even though coronary permeability increased. These results suggest that chronic ethanol consumption may mainly modulate the coronary haemodynamic function. However, ethanol did not reduce the damage to the coronary microvascular barrier to macromolecules caused by retrovirus infection.
In summary, altering coronary microvascular integrity may contribute to cardiovascular complications of AIDS. The structural and functional changes in the cardiovascular endothelium lead to increased coronary permeability to macromolecules. The retrovirus may directly or indirectly perturb the integrity of coronary endothelium. Chronic moderate ethanol consumption may be cardioprotective but did not reduce the structural destruction by the retrovirus.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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REFERENCES |
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Arbabi, S., Garcia, I., Bauer, G. J and Maier, R. V. (1999) Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway. Journal of Immunology 162, 74417445.
Barbaro, G., Di Lorenzo, G., Soldini, M., Giancaspro, G., Grisorio, B., Pellicelli, A. et al. (1999) Intensity of myocardial expression of inducible nitric oxide synthase influences the clinical course of human immunodeficiency virus-associated cardiomyopathy. Circulation 100, 933939.
Chen, G. J. and Watson, R. R. (1991) Modulation of tumor necrosis factor and gamma interferon production by cocaine and morphine in aging mice infected with LP-BM5, a murine retrovirus. Journal of Leukocyte Biology 50, 349355.[Abstract]
Chi, D., Henry, J., Kelley, J., Thorpe, R., Smith, J. K. and Krishnaswamy, G. (2000) The effects of HIV infection on endothelial function. Endothelium 7, 223242.[Medline]
Fitzpatrick, D. F., Hirschfield, S. L. and Coffey, R. G. (1993) Endothelium-dependent vasorelaxing activity of wine and other grape products. American Journal of Physiology 265, H774H778.
Floyd, R. A., Hensley, K., Jaffery, F., Maidt, L., Robinson, K., Pye, Q. et al. (1999) Increased oxidative stress brought on by pro-inflammatory cytokines in neurodegenerative processes and the protective role of nitrone-based free radical traps. Life Sciences 65, 18931899.[ISI][Medline]
Gelbard, H. A., Nottet, H. S., Swindells, S., Jett, M., Dzenko, K. A., Genis, P. et al. (1994) Platelet-activating factor: a candidate human immunodeficiency virus type 1-induced neurotoxin. Journal of Virology 68, 46284635.[Abstract]
Giese, N. A., Gazzinelli, R. T., Actor, J. K., Morawetz, R. A., Sarzotti, M. and Morse, H. C. (1996) Retrovirus-elicited interleukin-12 and tumor necrosis factor-alpha as inducers of interferon-gamma-mediated pathology in mouse AIDS. Immunology 87, 467474.[ISI][Medline]
Glass, J. D. and Johnson, R. T. (1996) Human immunodeficiency virus and the brain. Annual Review of Neuroscience 19, 126.[ISI][Medline]
Gu, J. W., Elam, J., Sartin, A., Li, W., Roach, R. and Adair, T. H. (2001) Moderate levels of ethanol induce expression of vascular endothelial growth factor and stimulate angiogenesis. American Journal of Physiology Regulatory Integrative and Comparative Physiology 281, R365R372.
Iso, H., Kitamura, A., Shimamoto, T., Sankai, T., Naito, Y., Sato, S. et al. (1995) Alcohol intake and the risk of cardiovascular disease in middle-aged Japanese men. Stroke 26, 767773.
Johann-Liang, R., Cervia, J. S. and Noel, G. J. (1997) Characteristics of human immunodeficiency virus-infected children at the time of death: an experience in the 1990s. Pediatric Infectious Disease Journal 16, 11451150.[ISI][Medline]
Lin, C. P., Lynch, M. C. and Kochevar, I. E. (2000) Reactive oxidizing species produced near the plasma membrane induce apoptosis in bovine aorta endothelial cells. Experimental Cell Research 259, 351359.[ISI][Medline]
McDonagh, P. F. (1983) Both protein and blood cells reduce coronary microvascular permeability to macromolecules. American Journal of Physiology 245, H698H706.[ISI][Medline]
McDonagh, P. F. and Williams, S. K. (1984) The preparation and use of fluorescentprotein conjugates for microvascular research. Microvascular Research 27, 1427.[ISI][Medline]
McKee, M. and Britton, A. (1998) The positive relationship between alcohol and heart disease in eastern Europe: potential physiological mechanisms. Journal of the Royal Society of Medicine 91, 402407.[Abstract]
Mollace, V., Nottet, H. S., Clayette, P., Turco, M. C., Muscoli, C., Salvemini, D. et al. (2001) Oxidative stress and neuroAIDS: triggers, modulators and novel antioxidants. Trends in Neurosciences 24, 411416.[ISI][Medline]
Pereira, C. F., Boven, L. A., Middel, J., Verhoef, J. and Nottet, H. S. (2000) Induction of cyclooxygenase-2 expression during HIV-1-infected monocyte-derived macrophage and human brain microvascular endothelial cell interactions. Journal of Leukocyte Biology 68, 423428.
Persidsky, Y., Zheng, J., Miller, D., Howard, E. and Gendelman, H. E. (2000) Mononuclear phagocytes mediate bloodbrain barrier compromise and neuronal injury during HIV-1-associated dementia. Journal of Leukocyte Biology 68, 413422.
Pinsky, D. J., Cai, B., Yang, X., Rodriguez, C., Sciacca, R. R. and Cannon, P. J. (1995) The lethal effects of cytokine-induced nitric oxide on cardiac myocytes are blocked by nitric oxide synthase antagonism or transforming growth factor beta. Journal of Clinical Investigation 95, 677685.[ISI][Medline]
Poikolainen, K., Vartiainen, E. and Korhonen, H. J. (1996) Alcohol intake and subjective health. American Journal of Epidemiology 144, 346350.[Abstract]
Redmond, E. M., Sitzmann, J. V. and Cahill, P. A. (2000) Potential mechanisms for cardiovascular protective effect of ethanol. Acta Pharmacologica Sinica 21, 385390.[ISI][Medline]
Ritter, L. S. and McDonagh, P. F. (1997) Low-flow reperfusion after myocardial ischemia enhances leukocyte accumulation in coronary microcirculation. American Journal of Physiology 273, H1154 H1165.
Schifitto, G., Sacktor, N., Marder, K., McDermott, M. P., McArthur, J. C., Kieburtz, K. et al. (1999) Randomized trial of the platelet-activating factor antagonist lexipafant in HIV-associated cognitive impairment. Neurological AIDS Research Consortium. Neurology 53, 391396.
Sei, Y., Nishida, K., Kustova, Y., Markey, S. P., Morse, H. C., 3rd and Basile, A. S. (1997) Pentoxifylline decreases brain levels of platelet activating factor in murine AIDS. European Journal of Pharmacology 325, 8184.[ISI][Medline]
Serradji, N., Bensaid, O., Martin, M., Kan, E., Dereuddre-Bosquet, N., Redeuilh, C. et al. (2000) Structureactivity relationships in platelet-activating factor (PAF). 10. From PAF antagonism to inhibition of HIV-1 replication. Journal of Medicinal Chemistry 43, 21492154.[ISI][Medline]
Stampfer, M. J., Colditz, G. A., Willett, W. C., Speizer, F. E. and Hennekens, C. H. (1988) A prospective study of moderate alcohol consumption and the risk of coronary disease and stroke in women. New England Journal of Medicine 319, 267273.[Abstract]
Syapin, P. J., Militante, J. D., Garrett, D. K. and Ren, L. (2001) Cytokine-induced iNOS expression in C6 glial cells: transcriptional inhibition by ethanol. Journal of Pharmacology and Experimental Therapeutics 298, 744752.
Van Tol, A. and Hendriks, H. F. (2001) Moderate alcohol consumption: effects on lipids and cardiovascular disease risk. Current Opinion in Lipidology 12, 1923.[ISI][Medline]
Wang, Y., Huang, D. S., Liang, B. and Watson, R. R. (1994) Nutritional status and immune responses in mice with murine AIDS are normalized by vitamin E supplementation. Journal of Nutrition 124, 20242032.[ISI][Medline]
Watson, R. R., Wang, J. Y., Dehghanpisheh, K., Huang, D. S., Wood, S., Ardestani, S. K. et al. (1995) T cell receptor V beta complementarity-determining region 1 peptide administration moderates immune dysfunction and cytokine dysregulation induced by murine retrovirus infection. Journal of Immunology 155, 22822291.[Abstract]
Welch, K. J. (2000) Correlates of alcohol and/or drug use among HIV-infected individuals. AIDS Patient Care and STDs 14, 317323.[ISI][Medline]
Westmoreland, S. V., Kolson, D. and Gonzalez-Scarano, F. (1996) Toxicity of TNF alpha and platelet activating factor for human NT2N neurons: a tissue culture model for human immunodeficiency virus dementia. Journal of Neurovirology 2, 118126.[ISI][Medline]
Xi, S., Cohen, D. and Chen, L. H. (1998) Effects of fish oil on cytokines and immune functions of mice with murine AIDS. Journal of Lipid Research 39, 16771687.
Zietz, C., Hotz, B., Sturzl, M., Rauch, E., Penning, R. and Lohrs, U. (1996) Aortic endothelium in HIV-1 infection: chronic injury, activation, and increased leukocyte adherence. American Journal of Pathology 149, 18871898.[Abstract]