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POLYAMINES: AN UNRECOGNISED CARDIOVASCULAR RISK FACTOR IN CHRONIC DIALYSIS?
412
be fully investigated before widespread mazine can be recommended.
use
of chlorpro-
J. H. is employed by the Swedish Medical Research Council, which supported his stay at the Cholera Research Laboratory. Requests for reprints should be addressed to G. H. R., I.C.D.D.R.B., G.P.O. 128, Dacca-2, Bangladesh. REFERENCES 1.
Rohde, J. E., Northrup, R. S. in Acute Diarrhoea in Childhood (edited by K. M. Elliot and J. Knight); Ciba Fdn Symp. 1976, 42, 336. 2. Field, M. ibid. p. 109. 3. Kakiuchi, S., Rall, T. W. Molec. Pharmac. 1968, 4, 367. 4. Wolf, J., Jones, A. B. Proc. natn. Acad. Sci. U.S.A. 1970, 65, 454. 5. Osnes, J. B., Christoffersen, T., Morland, J., Oye, I. Acta Pharmac. Toxicol. 1976, 38, 195. 6. 7. 8.
Lönnroth, I., Holmgren, J., Lange, S. Med. Biol. 1977, 55, 126. Holmgren, J., Lange, S., Lönnroth, I. Gastroenterology, 1978, 75, 1103. Andrén, B., Holmgren, J., Lange, S., Lönnroth, I., Martinsson, K. Unpub-
lished. 9. Benenson, A. S., Islam, M. R.,
Greenough, W. B., III, Bull. Wld Hlth Org. 1964, 30, 827. 10. King, M. Medical Care in Developing Countries. London, 1966. 11. Forrest, J. S., Carr, C. J., Usdin, E. (editors) Phenothiazines and Structually Related Drugs. New York, 1974. 12. Dupont, H. L., Hornick, R. B. J. Am. med. Ass. 1973, 226, 1525.
Hypothesis POLYAMINES: AN UNRECOGNISED CARDIOVASCULAR RISK FACTOR IN CHRONIC DIALYSIS?
JOHN D. BAGDADE
P. V. SUBBAIAH DAGMAR BARTOS FRANTISEK BARTOS ROBERT A. CAMPBELI
Department of Medicine, University of Washington School of Medicine, Providence Medical Center, Seattle, Washington, and
Department of Pediatrics, University of Oregon, Portland, Oregon, U.S.A. The
significance of raised polyamine (P.A.) levels in chronic-dialysis patients is unknown. Since these biologically active substances have hormone-like properties and promote cell-growth in plant and animal tissues, it is possible that they stimulate proliferation of arterial smooth-muscle cells (S.M.C.)—a central process in atherogenesis—and thereby contribute to the rapidly accelerated cardiovascular disease observed during dialysis. Such a role for P.A. is supported by tissue-culture studies, which show not only that P.A.—rich serum from dialysis patients stimulates S.M.C. growth, but also that this mitogenic effect is lost when P.A. are selectively removed from uræmic serum and restored by their addition. Although these observations provide new insights into possible mechanisms of atherogenesis, they are not surprising in view of the many known biological actions of P.A.
Summary
BACKGROUND
THE naturally occurring polyamines (P.A.) (putrescine, spermidine, and spermine) are found in all living cells. Since the biosynthesis and accumulation of these low-molecular-weight cationic compounds appear to be universal prerequisites of growth,l P.A. have been
of considerable interest to those engaged in cancer research. In general, however, their very existence and
relevance to human disease are little known to the clinician. Because of their apparently essential roles in the synthesis of R.N.A. and protein, the metabolism of cyclic nucleotides, and the stabilising of membrane-bound enzymes, P.A. influence a number of important cellular processes.3 In wound-healing, for example, and in compensatory hypertrophy and regeneration of stimulated or damaged tissues, the activity of polyamine-synthesising enzymes and cellular and circulating P.A. levels are all increased. Thus, during growth and replication, cellular putrescine and spermidine concentrations rise in sequence well before any change in R.N.A. is observed. As a result, whenever a peptide or steroid hormone (thyrotrophin, corticotrophin, growth hormone, cestrogens, androgens, corticosteroids) produces an anabolic response in its target tissue, an early stimulation in the activity of ornithine decarboxylase, the initiating and rate-limiting enzyme in P.A. synthesis, takes place. Alterations in P.A. metabolism appear to be important in the pathophysiology of a number of disorders in man. Normally excreted by the kidneys, P.A. rise sharply in renal failure.3 Because rapidly growing cells accumulate and release P.A., it is not surprising that P.A. levels are increased in blood and other tissues during pregnancy and in patients with cancer.4 Since blood-p.A. levels in cancer patients fluctuate with tumour activity, their measurement has been recommended as a clinical index of therapy. So potent and diverse are the biological actions of P.A. that it has been suggested that the uncanny similarity of many of the toxic gastrointestinal, neuromuscular, and haemopoietic manifestations of uraemia and widespread cancer may result from the accumulation of the same cationic peptide products of P.A.
degradation.33 While the growth-related properties of P.A. are obvious in cancer patients and during pregnancy, their mitogenic action is not immediately apparent clinically in the haemodialysis patient. Two observations suggest that P.A. may indeed promote tissue growth during chronic dialysis treatment in two undesirable ways: (i) dialysis patients develop cancer at an even higher frequency than renal allograft recipients5 and (ii) they also have a rapidly accelerated form of cardiovascular disease.6 Since cell-growth and replication are fundamental features of neoplasia and atherogenesis, P.A. might be unrecognised mitogens which promote both of these processes
in dialysis patients. EVIDENCE
Delayed renal excretion, abnormal membrane leakage, and probably suppression of a normal degradation pathway in renal failure contribute to the shift of P.A. from their normal intracellular location to excessively high levels in extracellular compartments. If P.A. are atherogenic in uraemia, then it is likely that they exert their deleterious effects on the intimal layer of the arterial wall, which is principally involved in atherosclerosis.’ The net effect of the yet unidentified immunological, hormonal, and toxic factor(s) in uraemia which disrupt the functional or structural integrity of the arterial endothelium and initiate atherogenesis is to increase the permeability of endothelium to substances in plasma, such as lipoproteins, hormones, P.A., and substances
413
released by the blood elements at injury sites. If any of these factors stimulate any one of the triad of processes that characterise all atherosclerotic lesions-s.M.c. proliferation, connective-tissue formation, or the deposition of lipid- then they are by definition atherogenic. Results of the following tissue-culture studies to assess the atherogenicity of serum from chronic-dialysis patients indicate that P.A. might influence the proliferative response of the arterial wall to injury. First, in a comparison of the mitogenic effects of pooled serum from dialysis patients and controls, both s.M.c. and fibroblasts grew faster in serum from dialysis patients (fig. 1).
selectively removed from this urxmic by passage through an activated silica-gel chromatographic column,8 a consistent reduction in mitogenicity of the serum was observed (fig. 2). This loss of activity, however, was restored by the addition of either the eluate from the column or the P.A., spermine. That a dose-dependent increase in cell growth was observed when spermine (one of the P.A. that is greatly elevated in urxmic serum) was added to control serum9 further supports the hypothesis that P.A. are important mitogens in the serum ofursemic patients.
When
P.A. were
serum
DISCUSSION
of premature cardiovascular disease without the well-recognised risks of hypertenpatients sion, diabetes, hyperlipidxmia, and cigarette smoking suggests that there are a number of other as yet unidentified factors which also promote atherogenesis. One example is homocystinxmia, an inborn error of metabolism in which high homocystine levels cause chronic endothelial injury,1O which presumably initiates the subsequent eventsin the arterial wall that lead to lesion formation. It seems likely that a similar form of toxic or immunologically mediated endothelial injury occurs chronically in uraemia and that this event is crucial in initiating lesion formation. Whether P.A. actually damage endothelium is not known, but the fact that these biologically active compounds and their metabolic congeners are known to affect adversely such important metabolic processes as trans-membrane glucose transport and the adenyl-cyclase and Na+-K+-A.T.p.ase enzyme systemsll suggests such a possibility. The
occurrence
in
Fig. 1—Effect of 10% serum pooled from chronic-dialysis patients on growth of human dermal fibroblasts and arterial smooth-muscle cells. Results
expressed as the mean percent of counts observed in four on days 5 and 10 of cells grown in 10% control serum.
experiments
Although
P.A.
are
implicated only theoretically
as
endothelial toxins in uraemia, our experiments strongly involve them as a stimulus for the s.M.c. proliferation that follows. Probably because homocystine can be converted to the P.A. precursor, methionine, methionine levels are increased in homocystingemic patients. Consequently, if their elevated methionine levels lead to the expected increase in P.A. levels, then the subintimal tissues of both homocystinxmic and chronic-dialysis patients may be exposed to high P.A. concentrations. If such pathways are operative, then altered P.A. metabolism may be one common biochemical denominator which accelerates atherogenesis in these two ostensibly dissimilar clinical disorders. This
study
was
supported by
National Institutes of Health grants
1-RO-HL-16219, l-R01-CA-16328-0382, and I-R01-HL22623 and grant from the M. J. Murdoch charitable trust:
a
Requests for reprints should be addressed to J. D. B., Providence Medical Center, 500-17th Avenue, Seattle, Washington 98124, U.S.A. REFERENCES
1. 2. 3.
Jänne, J., Pösö, H., Rama, A. Biochim. biophys. Acta, 1978, 473, 242. Jänne, J., Pösö, H., Raina, A. ibid. p. 246. Campbell, R. A., Talwalker, Y. B., Harner, M. H., et al. in Advances in Polyamme Research; vol. 2 (edited by R. A. Campbell, D. R. Morris, D. Bartos, G. D. Daves, and F. Bartos; p. 319. New York, 1978. 4. Marton, L. J., Russell, D. H., Levy, C. C. Clin. chim. Acta, 1973, 19, 926. 5. Matas, A. J., Simmons, R. L., Kjellstrand, C. M., Buselmeier, T. J., Najarian, J. S. Lancet, 1975, i, 883. 6. Lindner, A., Charra, B., Sherrard, D., Scnbner, B. New Engl. J. Med. 1974, 290, 697.
Fig.
2-Effects of removal of
polyamines from, and addition of
eluate and spermine (SPM, 50
dialysis patients
on
N.mol/1) to, serum of chronicthe growth of human arterial smooth-
muscle cells.
Results
expressed as percent (+P.A.).
nct urxmic serum
of cell
counts
observed
on
day
6 in in-
7. Ross, R., Glomset, J. A. ibid. 1976, 295, pp. 369, 420. 8. Grettie, D. P., Bartos, F., Bartos, D., Campbell, R. in Advances in Polyamine Research; vol. 2 (edited by R. A. Campbell, D. R. Morris, D. Bartos, G. D. Daves, and F. Bartos); p. 13. New York, 1978. 9. Bagdade, J. D., Campbell, D., Grettie, D. P., Bartos, D., Bartos, F. ibid. p. 346. 10. Harker, L. A., Slichter, S. J., Scott, C. R., Ross, R. New Engl. J. Med. 1974,
291, 537. 11.
Wright, R., Buehler, B. A., Rennert, O. M. Pediat. Res. 1976,10, 373.
be fully investigated before widespread mazine can be recommended.
use
of chlorpro-
J. H. is employed by the Swedish Medical Research Council, which supported his stay at the Cholera Research Laboratory. Requests for reprints should be addressed to G. H. R., I.C.D.D.R.B., G.P.O. 128, Dacca-2, Bangladesh. REFERENCES 1.
Rohde, J. E., Northrup, R. S. in Acute Diarrhoea in Childhood (edited by K. M. Elliot and J. Knight); Ciba Fdn Symp. 1976, 42, 336. 2. Field, M. ibid. p. 109. 3. Kakiuchi, S., Rall, T. W. Molec. Pharmac. 1968, 4, 367. 4. Wolf, J., Jones, A. B. Proc. natn. Acad. Sci. U.S.A. 1970, 65, 454. 5. Osnes, J. B., Christoffersen, T., Morland, J., Oye, I. Acta Pharmac. Toxicol. 1976, 38, 195. 6. 7. 8.
Lönnroth, I., Holmgren, J., Lange, S. Med. Biol. 1977, 55, 126. Holmgren, J., Lange, S., Lönnroth, I. Gastroenterology, 1978, 75, 1103. Andrén, B., Holmgren, J., Lange, S., Lönnroth, I., Martinsson, K. Unpub-
lished. 9. Benenson, A. S., Islam, M. R.,
Greenough, W. B., III, Bull. Wld Hlth Org. 1964, 30, 827. 10. King, M. Medical Care in Developing Countries. London, 1966. 11. Forrest, J. S., Carr, C. J., Usdin, E. (editors) Phenothiazines and Structually Related Drugs. New York, 1974. 12. Dupont, H. L., Hornick, R. B. J. Am. med. Ass. 1973, 226, 1525.
Hypothesis POLYAMINES: AN UNRECOGNISED CARDIOVASCULAR RISK FACTOR IN CHRONIC DIALYSIS?
JOHN D. BAGDADE
P. V. SUBBAIAH DAGMAR BARTOS FRANTISEK BARTOS ROBERT A. CAMPBELI
Department of Medicine, University of Washington School of Medicine, Providence Medical Center, Seattle, Washington, and
Department of Pediatrics, University of Oregon, Portland, Oregon, U.S.A. The
significance of raised polyamine (P.A.) levels in chronic-dialysis patients is unknown. Since these biologically active substances have hormone-like properties and promote cell-growth in plant and animal tissues, it is possible that they stimulate proliferation of arterial smooth-muscle cells (S.M.C.)—a central process in atherogenesis—and thereby contribute to the rapidly accelerated cardiovascular disease observed during dialysis. Such a role for P.A. is supported by tissue-culture studies, which show not only that P.A.—rich serum from dialysis patients stimulates S.M.C. growth, but also that this mitogenic effect is lost when P.A. are selectively removed from uræmic serum and restored by their addition. Although these observations provide new insights into possible mechanisms of atherogenesis, they are not surprising in view of the many known biological actions of P.A.
Summary
BACKGROUND
THE naturally occurring polyamines (P.A.) (putrescine, spermidine, and spermine) are found in all living cells. Since the biosynthesis and accumulation of these low-molecular-weight cationic compounds appear to be universal prerequisites of growth,l P.A. have been
of considerable interest to those engaged in cancer research. In general, however, their very existence and
relevance to human disease are little known to the clinician. Because of their apparently essential roles in the synthesis of R.N.A. and protein, the metabolism of cyclic nucleotides, and the stabilising of membrane-bound enzymes, P.A. influence a number of important cellular processes.3 In wound-healing, for example, and in compensatory hypertrophy and regeneration of stimulated or damaged tissues, the activity of polyamine-synthesising enzymes and cellular and circulating P.A. levels are all increased. Thus, during growth and replication, cellular putrescine and spermidine concentrations rise in sequence well before any change in R.N.A. is observed. As a result, whenever a peptide or steroid hormone (thyrotrophin, corticotrophin, growth hormone, cestrogens, androgens, corticosteroids) produces an anabolic response in its target tissue, an early stimulation in the activity of ornithine decarboxylase, the initiating and rate-limiting enzyme in P.A. synthesis, takes place. Alterations in P.A. metabolism appear to be important in the pathophysiology of a number of disorders in man. Normally excreted by the kidneys, P.A. rise sharply in renal failure.3 Because rapidly growing cells accumulate and release P.A., it is not surprising that P.A. levels are increased in blood and other tissues during pregnancy and in patients with cancer.4 Since blood-p.A. levels in cancer patients fluctuate with tumour activity, their measurement has been recommended as a clinical index of therapy. So potent and diverse are the biological actions of P.A. that it has been suggested that the uncanny similarity of many of the toxic gastrointestinal, neuromuscular, and haemopoietic manifestations of uraemia and widespread cancer may result from the accumulation of the same cationic peptide products of P.A.
degradation.33 While the growth-related properties of P.A. are obvious in cancer patients and during pregnancy, their mitogenic action is not immediately apparent clinically in the haemodialysis patient. Two observations suggest that P.A. may indeed promote tissue growth during chronic dialysis treatment in two undesirable ways: (i) dialysis patients develop cancer at an even higher frequency than renal allograft recipients5 and (ii) they also have a rapidly accelerated form of cardiovascular disease.6 Since cell-growth and replication are fundamental features of neoplasia and atherogenesis, P.A. might be unrecognised mitogens which promote both of these processes
in dialysis patients. EVIDENCE
Delayed renal excretion, abnormal membrane leakage, and probably suppression of a normal degradation pathway in renal failure contribute to the shift of P.A. from their normal intracellular location to excessively high levels in extracellular compartments. If P.A. are atherogenic in uraemia, then it is likely that they exert their deleterious effects on the intimal layer of the arterial wall, which is principally involved in atherosclerosis.’ The net effect of the yet unidentified immunological, hormonal, and toxic factor(s) in uraemia which disrupt the functional or structural integrity of the arterial endothelium and initiate atherogenesis is to increase the permeability of endothelium to substances in plasma, such as lipoproteins, hormones, P.A., and substances
413
released by the blood elements at injury sites. If any of these factors stimulate any one of the triad of processes that characterise all atherosclerotic lesions-s.M.c. proliferation, connective-tissue formation, or the deposition of lipid- then they are by definition atherogenic. Results of the following tissue-culture studies to assess the atherogenicity of serum from chronic-dialysis patients indicate that P.A. might influence the proliferative response of the arterial wall to injury. First, in a comparison of the mitogenic effects of pooled serum from dialysis patients and controls, both s.M.c. and fibroblasts grew faster in serum from dialysis patients (fig. 1).
selectively removed from this urxmic by passage through an activated silica-gel chromatographic column,8 a consistent reduction in mitogenicity of the serum was observed (fig. 2). This loss of activity, however, was restored by the addition of either the eluate from the column or the P.A., spermine. That a dose-dependent increase in cell growth was observed when spermine (one of the P.A. that is greatly elevated in urxmic serum) was added to control serum9 further supports the hypothesis that P.A. are important mitogens in the serum ofursemic patients.
When
P.A. were
serum
DISCUSSION
of premature cardiovascular disease without the well-recognised risks of hypertenpatients sion, diabetes, hyperlipidxmia, and cigarette smoking suggests that there are a number of other as yet unidentified factors which also promote atherogenesis. One example is homocystinxmia, an inborn error of metabolism in which high homocystine levels cause chronic endothelial injury,1O which presumably initiates the subsequent eventsin the arterial wall that lead to lesion formation. It seems likely that a similar form of toxic or immunologically mediated endothelial injury occurs chronically in uraemia and that this event is crucial in initiating lesion formation. Whether P.A. actually damage endothelium is not known, but the fact that these biologically active compounds and their metabolic congeners are known to affect adversely such important metabolic processes as trans-membrane glucose transport and the adenyl-cyclase and Na+-K+-A.T.p.ase enzyme systemsll suggests such a possibility. The
occurrence
in
Fig. 1—Effect of 10% serum pooled from chronic-dialysis patients on growth of human dermal fibroblasts and arterial smooth-muscle cells. Results
expressed as the mean percent of counts observed in four on days 5 and 10 of cells grown in 10% control serum.
experiments
Although
P.A.
are
implicated only theoretically
as
endothelial toxins in uraemia, our experiments strongly involve them as a stimulus for the s.M.c. proliferation that follows. Probably because homocystine can be converted to the P.A. precursor, methionine, methionine levels are increased in homocystingemic patients. Consequently, if their elevated methionine levels lead to the expected increase in P.A. levels, then the subintimal tissues of both homocystinxmic and chronic-dialysis patients may be exposed to high P.A. concentrations. If such pathways are operative, then altered P.A. metabolism may be one common biochemical denominator which accelerates atherogenesis in these two ostensibly dissimilar clinical disorders. This
study
was
supported by
National Institutes of Health grants
1-RO-HL-16219, l-R01-CA-16328-0382, and I-R01-HL22623 and grant from the M. J. Murdoch charitable trust:
a
Requests for reprints should be addressed to J. D. B., Providence Medical Center, 500-17th Avenue, Seattle, Washington 98124, U.S.A. REFERENCES
1. 2. 3.
Jänne, J., Pösö, H., Rama, A. Biochim. biophys. Acta, 1978, 473, 242. Jänne, J., Pösö, H., Raina, A. ibid. p. 246. Campbell, R. A., Talwalker, Y. B., Harner, M. H., et al. in Advances in Polyamme Research; vol. 2 (edited by R. A. Campbell, D. R. Morris, D. Bartos, G. D. Daves, and F. Bartos; p. 319. New York, 1978. 4. Marton, L. J., Russell, D. H., Levy, C. C. Clin. chim. Acta, 1973, 19, 926. 5. Matas, A. J., Simmons, R. L., Kjellstrand, C. M., Buselmeier, T. J., Najarian, J. S. Lancet, 1975, i, 883. 6. Lindner, A., Charra, B., Sherrard, D., Scnbner, B. New Engl. J. Med. 1974, 290, 697.
Fig.
2-Effects of removal of
polyamines from, and addition of
eluate and spermine (SPM, 50
dialysis patients
on
N.mol/1) to, serum of chronicthe growth of human arterial smooth-
muscle cells.
Results
expressed as percent (+P.A.).
nct urxmic serum
of cell
counts
observed
on
day
6 in in-
7. Ross, R., Glomset, J. A. ibid. 1976, 295, pp. 369, 420. 8. Grettie, D. P., Bartos, F., Bartos, D., Campbell, R. in Advances in Polyamine Research; vol. 2 (edited by R. A. Campbell, D. R. Morris, D. Bartos, G. D. Daves, and F. Bartos); p. 13. New York, 1978. 9. Bagdade, J. D., Campbell, D., Grettie, D. P., Bartos, D., Bartos, F. ibid. p. 346. 10. Harker, L. A., Slichter, S. J., Scott, C. R., Ross, R. New Engl. J. Med. 1974,
291, 537. 11.
Wright, R., Buehler, B. A., Rennert, O. M. Pediat. Res. 1976,10, 373.