- Email: [email protected]
Density-dependent contraction of the endothelial nascent histamine pool by exogenous heparin
EXPERIMENTAL
AND
MOLECULAR
PATHOLOGY
45,93- 102 (1986)
Density-Dependent Contraction of the Endothelial Histamine Pool by Exogenous Heparinl RANDAL S.BLANKANDTHEODORE 208 Mueller
Laboratory,
Received
M. HOLLIS~
Department of Biology, The Pennsylvania University Park, Pennsylvania 16802
November
22. 1985, and in revisedform
Nascent
March
State
II.
University,
1986
The aortic endothelial cell nascent histamine pool has been implicated in the control of vessel wall permeability under conditions of stress and injury. We report the contraction of this histamine pool in low density bovine aortic endothelial cell (BAEC) cultures by exogenous heparin. Untreated BAEC exhibit a decline in histamine content in 3-day cultures with increasing plating density between 1000 and 16,000 cells/cm*. Heparin abolished this density-related difference by effecting a 67% contraction of the histamine pool in the lowest density cultures. This effect was reversible and specific to heparin. At a confluent density, endothelial cells secrete heparin-like glycosaminoglycans which affect smooth muscle and endothelial metabolism. We propose that the metabolic effects of exogenous heparin, and perhaps endogenous heparins. extend to specific modulations of the BAEC nascent histamine pool. @ 1986 Academic Press, Inc.
INTRODUCTION The heparin-like compounds comprise a class of glycosaminoglycans which have been recognized for some time as anticoagulants present in anaphylactic shock (Jaques and Waters, 1941; Engleberg, 1977). Recently a body of evidence has accrued which ascribes to them important functions in the regulation of vascular cell metabolism, growth, and maintenance of endothelial integrity. Castellot et ul. (1981) identified the presence of a heparin or heparin-like molecule in primary cultures of endothelial cells which inhibits smooth muscle cell proliferation in vitro. The involvement of heparin in the control of smooth muscle cell (SMC) growth has also been demonstrated in vivo (Clowes and Karnovsky, 1977; Guyton et al., 1980; Clowes and Clowes, 1984). Guyton et al. (1980) and Clowes and Clowes (1984) have shown that heparin administration following arterial desiccation and balloon injury inhibits myointimal thickening in rat arteries. In addition to its effects on arterial smooth muscle, heparin exerts potentially important effects at the level of the vascular endothelial cell surface. Glimelius et al. (1978) and Hiebert and Jaques (1976) have confirmed that the binding of 3Hheparin to the surface of cultured human endothelial cells is saturable, reversible, trypsin-sensitive, and unaffected by large amounts of other glycosaminoglycans. The binding of heparins to their endothelial cell receptors, in addition to eliciting a number of cellular responses, may confer specific properties at these sites. These include effects on local antithrombotic properties (Pearson, 1983), lipoprotein-lipase binding (Williams et al., 1983), and prostacyclin activity (Baluda et al., 1982). Endogenous heparin sulfate, a related compound, also participates in these reactions and as such may be considered antagonistic to the development of atherosclerotic lesions (Berenson et al., 1985). r Supported by the Coronary Heart Disease Research Project, a Program of the American Health Assistance Foundation. z To whom requests for reprints should be addressed. 93 OOl4-4800/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
94
BLANK
AND
HOLLIS
The arterial nascent histamine pool has been implicated in the regulation of vessel wall permeability under conditions of stress and injury. In view of the anti-atherogenic nature of heparin sulfates (Berenson et al., 1985) and previous work demonstrating the effects of exogenous heparin on metabolic pathways for histamine (Hahn et al., 1966), this study has been undertaken to determine the effects of heparin on the nascent histamine pool of aortic endothelial and smooth muscle cells with respect to cell density. We report here the contraction of this histamine pool in low density aortic endothelial cell cultures by exogenous heparin. MATERIALS
AND METHODS
Cell culture. Endothelial cells were isolated from the thoracic aorta of mature bovine females. Aortas were aseptically excised from the animal immediately following slaughter, placed in ice-cold sterile Hanks’ balanced salt solution (HBSS) with 100 U/ml penicillin-streptomycin (Flow Laboratories, McLean, Va.) After a rinse in sterile HBSS, the aortas were cleaned of adventitia and given a final rinse in calcium and magnesium-free HBSS. One end of the aortic segment was clamped with large hemostats, the intercostals with sterile seraphines. A volume of 0.1% collagenase-dispase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and 0.15% trypsin (Sigma Chemical Co., St. Louis, MO.) in calcium and magnesium-free HBSS sufficient to fill the lumenal space was pipetted through the open end of the aortic segment. The closed aorta was incubated for approximately 1.5min at 37°C in 5% CO, and agitated by gentle rolling during this period. The aorta was opened and the resultant cell suspension pipetted in lo-ml aliquots to 15-ml centrifuge tubes containing 5 ml Minimum Essential Medium (MEM, Flow Laboratories) supplemented with 10% fetal bovine serum (Sterile Systems, Logan, Utah) to inhibit further proteolytic activity. Each tube was centrifuged for 5 min, the supernatant discarded, the pellet resuspended in the serum-supplemented MEM and plated at 50,000 cells/ml in 75-cm* tissue culture flasks (Costar, Cambridge, Mass,). Cultures were then incubated with 5% CO2 in air and grown to confluency. Bovine aortic smooth muscle cells were the generous gift of Dr. Patricia D’Amore and Dr. Alicia Orlidge of Children’s Hospital, Boston. Endothelial and smooth muscle cells were plated on plastic 35-mm petri dishes at 1000, 4000, or 16,000 cells/cm2. After a 4-hr period for cell attachment, cultures were treated with their respective concentration of protamine, hyaluronic acid (Sigma), chondroitin sulfate (Miles Scientific, Naperville, Ill.), or heparin (Hepar Industries, Franklin, Ohio) and incubated in its presence for 72 hr. Since glycosaminoglycan treatment can be expected to affect the growth of cells in culture, we have chosen, for the sake of uniformity, to express results in terms of the initial plating density. Final cell densities under all tested culture conditions were recorded and are presented in the text. Preparation of endothelial and smooth muscle cells for assaying histamine content and histidine decarbo-;ylase activity. Following the experimental period, cell cultures were lysed and collected at 0°C in 0.5 ml of cold 0.1 M phosphatebuffered saline (PBS, pH 7.9) containing 0.1% Triton X-100. Samples were aliquoted in 0.5-ml volumes, frozen immediately, and stored at - 80°C. Determination of histidine decarboxylase activity and histamine content. These parameters were determined using a modification of the technique of
EFFECTS OF HEPARIN
ON ENDOTHELIAL
CELL HISTAMINE
95
6
1X10’ PLATING
4x 103 DENSITY (cells/cm2
I 1
FIG. 1. Histamine content and HD activity as a function of plating density in 3-day cultures of BAEC. Values shown represent means f standard error.
Taylor and Snyder (1972) as previously described (Orlidge and Hollis, 1982). Briefly, the N-methylation of histamine was carried out in the presence of [methyf-3H]SAMe and histamine methyltransferase solution prepared from rat kidneys. Following incubation, samples were boiled and subjected to two extractions in toluene and isoamyl alcohol with intervening desiccation. Samples were counted on a Packard Tri-Carb scintillation counter. Counts in disintegrations per minute were regressed against known standards for the determination of histamine parameters. Histamine content was determined from samples incubated in D-histidine, and histidine decarboxylase activity, from samples incubated with L-histidine. Standard curves with and without 10e4 g/ml were compared to ensure that residual heparin from the cell lysate did not interfere with the N-methylation of histamine. Final histidine decarboxylase activity was expressed as nanograms of histamine formed per 100,000 cells per hour and histamine content as nanograms per 100,000 cells. Data were analyzed by one factor analysis of variance. Significance by this test was followed by appropriate tests for multiple comparisons. Group means with equal sample number were compared using Duncan’s multiple range test. The Newman-Keuls test for multiple comparison was used to compare means from groups unequal in sample number. RESULTS Untreated endothelial cell cultures exhibit a density-related decline in histamine content (Fig. 1). This fall in cellular histamine content amounts to a 94% decline between cultures plated at 16,000 cells/cm2 and those plated at 1000 cells/ cm2. Histidine decarboxylase activity, on the other hand, declines insignificantly with an increase in the plating density of 3-day cultures (Fig. 1). Figure 2 depicts the effects of 10e7 g/ml heparin on histamine content in endothelial cell cultures over the same range of plating densities. The effects of exogenous heparin on this parameter is significant only at the low plating density, where a 67% contraction of the nascent histamine pool is observed. While effecting a drop in endothelial histamine content, heparin treatment resulted in a density-dependent and dose-related stimulation of histidine decarboxylase (HD) activity (Fig. 3). The overall level of HD activity and the degree of
96
BLANK
:t T
AND HOLLIS
\
Ix IO’ PLATING
4x10’ DENSITY (cells/cm’
I I
,6x10’
)
FIG. 2. Effects of heparin (IO-’ g/ml) on histamine content in BAEC cultures plated at varying
stimulation by heparin treatment was greatest in subconfluent cultures of endothelium and progressively less as plating density was increased. Somewhat surprisingly, the activity of HD does not parallel the heparin-induced contraction of the histamine pool (Fig. 4). While histamine content falls about 67% with heparin treatment, HD activity is stimulated in a dose-related manner up to almost fivefold at the highest concentration tested. Heparin at 10T7 g/ml was as effective as higher concentrations in causing the decrease in histamine content. Untreated aortic SMC also exhibit a density-related decline in histamine content (Fig. 5). However, the response of the SMC histamine pool to heparin treatment is decidedly different from that of the bovine aortic endothelial cell (BAEC) (Fig. 6). First, the absolute level of histamine in control BAEC cultures is more than twice that of control SMC. Second, BAEC responds to heparin treatment by a 67% contraction of the nascent histamine pool. SMC do not exhibit any significant change in this parameter over the range of heparin concentrations tested. However, in low density SMC cultures, heparin in concentrations between lop7 and 1O-4 g/ml also stimulated HD activity in a dose-related manner. At 1O-4
FIG, 3. Dose-response
curve depicting the effects of heparin on HD activity at varying plating
EFFECTS
OF HEPARIN
0
ON ENDOTHELIAL
16’ HEPARIN
16” DOSAGE
CELL
HISTAMINE
Id’
97
Id’
(g /ml)
FIG. 4. Comparison of the dose-response curves for both HD activity and histamine content with heparin treatment in low density BAEC cultures.
g/ml this represented a fivefold increase over control values. Again, activity of HD does not correlate well with the histamine curve, suggesting the significance of compensating metabolic processes (Fig. 7). In further experiments, the effects of protamine sulfate on heparin-induced alterations in histamine content were examined. Again, treatment with heparin caused a greater than 50% drop in cellular histamine content (Fig. 8). Protamine sulfate (lO-‘j g/ml) completely reversed the effects of heparin on this parameter, while protamine sulfate alone did not alter histamine content significantly from the control or from either of the treatments. This effect of heparin is not only inhibitable, but it is relatively specific in that chondroitin sulfate and hyaluronic acid failed to effect a significant contraction of the BAEC nascent histamine pool (Fig. 9). Finally, 72-hr culture densities for BAEC and SMC cultures are reported in Tables 1 and 2, respectively. These data demonstrate that final cell densities parallel the initial plating density and suggest that neither BAEC nor SMC cultures plated at 1000 cells/cm2 are at a proliferative disadvantage relative to those plated
‘t
i’
\
1-d IX IO'
PLATING
4x IO’ DENSITY (cells/cm*
I6.10* )
FIG. 5. Histamine content and HD activity as a function of plating density in 3-day cultures of SMC.
BLANK
AND HOLLIS
.. 6 ..5
I 10.’
0
HEPARIN
FIG. 6. Comparison of the dose-response cultures with heparin treatment.
IODOSAGE
1 g/ml
10.’ 1
16’
curves for histamine content between BAEC and SMC
at higher densities, though some of the reagents tested, heparin and hyaluronic acid, have dramatic effects on final cell density. DISCUSSION It is generally agreed that the vascular endothelium serves as a permeability barrier against lumenal materials entering the arterial wall. Most of the mammalian arterial wall is avascular and depends on bloodborne materials for nutrition. The endothelium must then regulate the type and amount of molecules passing through the intimal layer. Furthermore, it is believed that disruption of the endothelial barrier, though perhaps not obligatory, nevertheless contributes to intimal thickening via the subsequent interaction of growth factors with underlying smooth muscle cells (Ross, 1986). Disruption of the endothelial barrier occurs with all types of endothelial injury and is accompanied by increases in vessel wall permeability. Aortic histamine
I 0
I Id’
lo-” HEPPiRlN
DOSAGE
16’
16’
(g/ml)
FIG. 7. Effects of varying doses of heparin on histamine content and HD activity in low density SMC cultures.
EFFECTS OF HEPARIN
ON ENDOTHELIAL
FIG. 8. Effects of protamine sulfate on the heparin-induced density BAEC cultures.
CELL HISTAMINE
99
contraction of the histamine pool in low
metabolism is altered in a variety of atherogenic risk situations, including diabetes (Orlidge and Hollis, 1982; Gallik and Hollis, 1981), hypercholesterolemia (Markle and Hollis, 1975; Hollis and Sloss, 1975), and shear stress (DeForrest and Hollis, 1980; Paraschos and Hollis, 1985). Orlidge and Hollis (1982) reported that in streptozotocin-diabetic rats, aortic endothelial cell histamine content increased 138%, HD activity increased 250%, and histaminase activity decreased 50% over the 4-week holding period. In the underlying smooth muscle the histamine content increased over 150% and the HD activity increased over 300% with respect to control values. Insulin treatment completely reversed these metabolic alterations. Other work has also indicated that the histamine-forming capacity (HFC) of the aorta is increased in cholesterol-fed rabbits and that this increased HFC is intimately associated with an increased uptake into the arterial wall (Owens and Hollis, 1979; Hollis and Furniss, 1980). Owens and Hollis (1979) demonstrated that the partial inhibition of HD activity in New Zealand White rabbits resulted in a 51% reduction in aortic albumin up-
FIG. 9. The effects of chondroitin cultures plated at low density.
sulfate and hyaluronic acid on the histamine content of BAEC
100
BLANK
AND HOLLIS
TABLE 1 Final BAEC Densities in Cultures Plated at 1000. 4000, and 16,000 Cells/cm*
Treatment Control Heparin
Chondroitin sulfate Hyaluronic acid Protamine sulfate Protamine sulfate + heparin
Concentration kidml) IO-’ IO-6 10-S 10-4 10-4 10-4 IO-6 10-6 10-d
Initial plating density (cells/cm*) 1000
4000
16,000
1.14 0.97 0.70 0.71 1.00 1.09 0.65 1.27 0.89
3.73 4.32 3.60 3.57 3.68 -
8.85 8.78 9.12 8.68 9.42 -
Note. Only cultures plated at the lowest density were treated with chondroitin sulfate (10m4 g/ml), hyaluronic acid ( 1O-4 g/ml), and protamine sulfate ( 10e6 g/ml) for comparison to the effects of heparin. Higher density cultures were unresponsive to heparin treatment.
take and a 63% reduction in oil red 0 staining in thoracic aortas from rabbits fed a cholesterol diet. Later, Owens and Hollis (1981), using Evans blue dye to identify areas of spontaneous arterial injury in canine aortas, found a significant correlation between local albumin accumulation, HD activity, and histamine content. There are other reports as well of an expansion of this nascent histamine pool in hypertension (Bolitho and Hollis, 1975). Data from this laboratory have also confirmed an increase in the histamine content of cultured BAEC subjected to elevated shear stress (Paraschos and Hollis, 1985). Finally, Kalsner and Richards (1984) have found marked increases in the histamine content of coronary arteries from hearts of cardiac patients and in vessel segments assessed as atherosclerotic. While the relationship between the histamine content of various tissues and conditions of injury are well established, specific effects of this nascent histamine pool expansion are less well understood. Both BAEC and SMC exhibit a dramatic density-dependent decline in histamine content. Higher density cultures of both cell types are relatively depleted of this amine. In view of the data presented here, it appears that the size of the nascent pool may be related directly or indirectly to the rate of cell division and turnover. Indeed, exogenous histamine has been shown to stimulate the growth of nonquiescent BAEC (D’Amore and Shepro, 1977). Experimental conditions such as hypercholesterolemia which induce an expansion of the aortic histamine pool (Hollis and Furniss, 1980; Owens and Hollis, 1979) also predispose the endothelium to injury or possible desquamation (Massman and Jellinek, 1980). In either case, the increase in cell turnover under these conditions may be associated with an acute expansion of the histamine pool and resultant alterations in transmural permeability. The effects of histamine on decreasing actin cables in endothelium may, in part, explain the action of this amine in inflammatory permeability (Welles et al., 1985). It is of interest that in this study contraction of the BAEC histamine pool was observed only in cultures plated at low density. At a confluent density, endothelial cells secrete heparin-like glycosaminoglycans which affect SMC and BAEC metabolism (Fritze et al., 1985; Gadjusek, 1985). That the density-related decline
EFFECTS OF HEPARIN
ON ENDOTHELIAL
TABLE 11 Final SMC Densities in Cultures Treated with Heparin (lo-’
Control Heparin
to 10e4 g/ml)
Initial plating density (cells/cm*)
Concentration (g/ml)
Treatment
101
CELL HISTAMINE
10-7 IO-6 10-s IO-4
1000
4000
16.000
1.43 1.38 0.85 0.75 0.57
5.38 4.98 4.71 3.50 I .93
13.00 11.63 11.40 9.90 7.95
in BAEC histamine content may be associated with a concomitant rise in endogenous heparins is suggested by the failure of exogenous heparin to affect this parameter in cultures plated at higher densities. The control point of the putative regulation has not been elucidated in this study. However, it appears that heparin may regulate the overall level of the histamine pool by modulating histamine catabolism. This is suggested indirectly by several lines of evidence. First, heparin stimulates activity of the synthetic enzyme in both SMC and BAEC in a dose-related manner, while effecting a histamine pool contraction only in BAEC cultures. Second, the density-related decline in histamine content in untreated cultures of BAEC and SMC appears to be independent of changes in the activity of HD. Finally, heparin has been shown to enhance catabolic pathways for histamine in other systems (Hahn et al., 1966). ACKNOWLEDGMENTS The authors thank Robert Sapanski, Charles Smith, and Mark Schmidt for their invaluable technical assistance and Dr. Patricia D’Amore and Dr. Alicia Orlidge for their generous gift of smooth muscle cells. We are also indebted to Mary Alice Shea for the typing of this manuscript.
REFERENCES BALUDA. V. I?, SUSHKEVICH. G. N., and LIASKO, L. I. (1982). Effect of heparin on prostacychn activity. Biul. Eksp. Biol. Med. 94, 79-80. BERENSON, G. S.. RADHAKRISHNAMURTHY. B.. SRINIVASAN, S. R., VIJAYAGOPAL, P., and DALFERES. E. R. (1985). Proteoglycans and potential mechanisms related to atherosclerosis. Ann. N. Y. Acad.
Sci. 454, 69-78.
BOLITHO. G. A., and HOLLIS, T. M. (1975). Aortic histamine synthesis in experimental hypertension. Proc.
Sot.
Exp.
Biol.
Med.
148, I I89-
I 192.
CASTELLOT, J. J., JR.. ADDONIZIO. M. L., ROSENBERG. R., and KARNOVSKY, M. J. (1981). Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth. J. Cell Biol. 90, 372-379. CLOWES. A. W., and CLOWES. M. M. (1984). Heparin inhibits injury-induced arterial smooth muscle migration and proliferation. Fed. Proc. 43, 459. CLOWES, A. W.. and KARNOVSKY, M. J. (1977). Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nrrtlrre (London) 265, 625-626. D’AMORE, P., and SHEPRO, D. (1977). Stimulation of growth and calcium influx in cultured bovine aortic endothelial cells by platelets and vasoactive substances. J. Cell Physiol. 92, 177- 184. DEFOREST, J. M., and HOLLIS. T. M. (1980). Relationship between low intensity shear stress, aortic histamine formation, and aortic albumin uptake. Exp. Mol. Putho/. 32, 217-225. ENGLEBERG, H. (1977). Probable physiological functions of heparin. Fed. Proc. 36, 70-72. FRITZE, L. M. S., REILLY, C. F., and ROSENBERG, R. (1985). An antiproliferative heparin sulfate species produced by postconfluent smooth muscle cells. J. Cell Biol. 100, 1041- 1049.
102
BLANK
AND
HOLLIS
GADJUSEK, C. M. (1985). Heparin release of endothelial cell-derived mitogens. Fed. Proc. 44, 1655. GALLIK, S., and HOLLIS, T. M. (1981). Studies on aortic histamine synthesis in experimental diabetes. Proc. Sot. Exp. Biol. Med. 166, 496-500. GLIMELIUS, B., BUSCH, C., and HOOK, M. (1978). Binding of heparin on surface of cultured human endothelial cells. Thromb. Res. 12, 773-782. GUYTON, J. R., ROSENBERG, R. D., CLOWES, A. W., and KARNOVSKY, M. J. (1980). Inhibition of rat arterial smooth muscle cell proliferation by heparin. Circ. Res. 46, 625-634. HAHN, F., SCHMUTZLER, W., SESEKE, G., GIERTZ, H., and BERNAUER. W. (1966). Histaminasefreisetzung durch heparin und protamin beim meerschweinchen. Biochem. Pharmacol. 15, 155-160. HIEBERT, L. M., and JAQUES, L. B. (1976). Heparin uptake on endothelium. Artery 2, 26-37. HOLLIS. T. M., and FURNISS, J. V. (1980). Relationship between aortic histamine formation and aortic albumin permeability in atherogenesis. Proc. Sot. Exp. Biol. Med. 165, 271-274. HOLLIS, T. M., and SLOSS, R. J. (1975). Rabbit aortic histamine synthesis following short term cholesterol feeding. Atherosclerosis 21, 125-134. JAQUES, L. B., and WATERS, L. B. (1941). The identity and origin of the anti-coagulant of anaphylactic shock in the dog. .I. Physiol. 99, 454-466. KALSNER, J., and RICHARDS, R. (1984). Coronary arteries of cardiac patients are hyperreactive and contain stores of amines: A mechanism for coronary spasm. Science 223, 143.5-1437. MARKLE, R. A., and HOLLIS. T. M. (1975). Rabbit aortic endothelial and medial histamine synthesis following short-term cholesterol feeding. Esp. Mol. Puthol. 23, 417-426. MASSMAN, J.. and JELLINEK, H. (1980). Hematogenic infiltration of the aortic intima in normal and hypercholesterolemic swine: Studies on en face endothelium-intima preparations. Exp. Mol. Parhol.
18, 1 I-24.
ORLIDGE. A., and HOLLIS, T. M. (1982). Aortic endothelial and smooth muscle histamine metabolism in experimental diabetes. Arferiosclereosis 21, 125- 134. OWENS, G. K., and HOLLIS, T. M. (1979). Relationship between inhibition of aortic histamine formation, aortic albumin permeability and atherosclerosis. Atherosclerosis 34, 365-373. OWENS, G. K., and HOLLIS, T. M. (1981). Local aortic histamine metabolism and albumin accumulation: differences between blue and white areas. Arrheriosclerosis 1, 265-272. PARASCHOS, S. I., and HOLLIS, T. M. (1985). Effects of shear stress on bovine endothelial cells, in vitro, with relation to the pathogenesis of atherosclerosis. Fed. Proc. 44, 1659. PEARSON, J. D. (1983). Endothelial cell metabolism. Prog. Appl. Microcirc. 1, Il4- 126. ROSS, R. (1986). The pathogenesis of atherosclerosis-an update. N. Engl. J. Med. 314, 488-500. SCHWARTZ, S., GADJUSEK, C., REIDY, M.. SELDEN, S., III, and HAUDENSCHILD. C. (1980). Maintenance of integrity in aortic endothelium. Fed. Proc. 39, 2618-2624. TAYLOR, K. M., and SNYDER, S. H. (1972). Dynamics of the regulation of histamine levels in mouse brain. J. Neurochem. 19, 341-354. WELLES, S. L., SHEPRO, D., and HECHTMAN, H. B. (1985). Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium. J. Cell. Physiol. 123, 337-342. WILLIAMS, M. P., STREETER, H. B., WUSTEMAN. F. S., and CRYER, A. (1983). Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium. Biochim. Biophys. Acta 761, 83-91.
AND
MOLECULAR
PATHOLOGY
45,93- 102 (1986)
Density-Dependent Contraction of the Endothelial Histamine Pool by Exogenous Heparinl RANDAL S.BLANKANDTHEODORE 208 Mueller
Laboratory,
Received
M. HOLLIS~
Department of Biology, The Pennsylvania University Park, Pennsylvania 16802
November
22. 1985, and in revisedform
Nascent
March
State
II.
University,
1986
The aortic endothelial cell nascent histamine pool has been implicated in the control of vessel wall permeability under conditions of stress and injury. We report the contraction of this histamine pool in low density bovine aortic endothelial cell (BAEC) cultures by exogenous heparin. Untreated BAEC exhibit a decline in histamine content in 3-day cultures with increasing plating density between 1000 and 16,000 cells/cm*. Heparin abolished this density-related difference by effecting a 67% contraction of the histamine pool in the lowest density cultures. This effect was reversible and specific to heparin. At a confluent density, endothelial cells secrete heparin-like glycosaminoglycans which affect smooth muscle and endothelial metabolism. We propose that the metabolic effects of exogenous heparin, and perhaps endogenous heparins. extend to specific modulations of the BAEC nascent histamine pool. @ 1986 Academic Press, Inc.
INTRODUCTION The heparin-like compounds comprise a class of glycosaminoglycans which have been recognized for some time as anticoagulants present in anaphylactic shock (Jaques and Waters, 1941; Engleberg, 1977). Recently a body of evidence has accrued which ascribes to them important functions in the regulation of vascular cell metabolism, growth, and maintenance of endothelial integrity. Castellot et ul. (1981) identified the presence of a heparin or heparin-like molecule in primary cultures of endothelial cells which inhibits smooth muscle cell proliferation in vitro. The involvement of heparin in the control of smooth muscle cell (SMC) growth has also been demonstrated in vivo (Clowes and Karnovsky, 1977; Guyton et al., 1980; Clowes and Clowes, 1984). Guyton et al. (1980) and Clowes and Clowes (1984) have shown that heparin administration following arterial desiccation and balloon injury inhibits myointimal thickening in rat arteries. In addition to its effects on arterial smooth muscle, heparin exerts potentially important effects at the level of the vascular endothelial cell surface. Glimelius et al. (1978) and Hiebert and Jaques (1976) have confirmed that the binding of 3Hheparin to the surface of cultured human endothelial cells is saturable, reversible, trypsin-sensitive, and unaffected by large amounts of other glycosaminoglycans. The binding of heparins to their endothelial cell receptors, in addition to eliciting a number of cellular responses, may confer specific properties at these sites. These include effects on local antithrombotic properties (Pearson, 1983), lipoprotein-lipase binding (Williams et al., 1983), and prostacyclin activity (Baluda et al., 1982). Endogenous heparin sulfate, a related compound, also participates in these reactions and as such may be considered antagonistic to the development of atherosclerotic lesions (Berenson et al., 1985). r Supported by the Coronary Heart Disease Research Project, a Program of the American Health Assistance Foundation. z To whom requests for reprints should be addressed. 93 OOl4-4800/86 $3.00 Copyright 0 1986 by Academic Press. Inc. All rights of reproduction in any form reserved.
94
BLANK
AND
HOLLIS
The arterial nascent histamine pool has been implicated in the regulation of vessel wall permeability under conditions of stress and injury. In view of the anti-atherogenic nature of heparin sulfates (Berenson et al., 1985) and previous work demonstrating the effects of exogenous heparin on metabolic pathways for histamine (Hahn et al., 1966), this study has been undertaken to determine the effects of heparin on the nascent histamine pool of aortic endothelial and smooth muscle cells with respect to cell density. We report here the contraction of this histamine pool in low density aortic endothelial cell cultures by exogenous heparin. MATERIALS
AND METHODS
Cell culture. Endothelial cells were isolated from the thoracic aorta of mature bovine females. Aortas were aseptically excised from the animal immediately following slaughter, placed in ice-cold sterile Hanks’ balanced salt solution (HBSS) with 100 U/ml penicillin-streptomycin (Flow Laboratories, McLean, Va.) After a rinse in sterile HBSS, the aortas were cleaned of adventitia and given a final rinse in calcium and magnesium-free HBSS. One end of the aortic segment was clamped with large hemostats, the intercostals with sterile seraphines. A volume of 0.1% collagenase-dispase (Boehringer Mannheim Biochemicals, Indianapolis, Ind.) and 0.15% trypsin (Sigma Chemical Co., St. Louis, MO.) in calcium and magnesium-free HBSS sufficient to fill the lumenal space was pipetted through the open end of the aortic segment. The closed aorta was incubated for approximately 1.5min at 37°C in 5% CO, and agitated by gentle rolling during this period. The aorta was opened and the resultant cell suspension pipetted in lo-ml aliquots to 15-ml centrifuge tubes containing 5 ml Minimum Essential Medium (MEM, Flow Laboratories) supplemented with 10% fetal bovine serum (Sterile Systems, Logan, Utah) to inhibit further proteolytic activity. Each tube was centrifuged for 5 min, the supernatant discarded, the pellet resuspended in the serum-supplemented MEM and plated at 50,000 cells/ml in 75-cm* tissue culture flasks (Costar, Cambridge, Mass,). Cultures were then incubated with 5% CO2 in air and grown to confluency. Bovine aortic smooth muscle cells were the generous gift of Dr. Patricia D’Amore and Dr. Alicia Orlidge of Children’s Hospital, Boston. Endothelial and smooth muscle cells were plated on plastic 35-mm petri dishes at 1000, 4000, or 16,000 cells/cm2. After a 4-hr period for cell attachment, cultures were treated with their respective concentration of protamine, hyaluronic acid (Sigma), chondroitin sulfate (Miles Scientific, Naperville, Ill.), or heparin (Hepar Industries, Franklin, Ohio) and incubated in its presence for 72 hr. Since glycosaminoglycan treatment can be expected to affect the growth of cells in culture, we have chosen, for the sake of uniformity, to express results in terms of the initial plating density. Final cell densities under all tested culture conditions were recorded and are presented in the text. Preparation of endothelial and smooth muscle cells for assaying histamine content and histidine decarbo-;ylase activity. Following the experimental period, cell cultures were lysed and collected at 0°C in 0.5 ml of cold 0.1 M phosphatebuffered saline (PBS, pH 7.9) containing 0.1% Triton X-100. Samples were aliquoted in 0.5-ml volumes, frozen immediately, and stored at - 80°C. Determination of histidine decarboxylase activity and histamine content. These parameters were determined using a modification of the technique of
EFFECTS OF HEPARIN
ON ENDOTHELIAL
CELL HISTAMINE
95
6
1X10’ PLATING
4x 103 DENSITY (cells/cm2
I 1
FIG. 1. Histamine content and HD activity as a function of plating density in 3-day cultures of BAEC. Values shown represent means f standard error.
Taylor and Snyder (1972) as previously described (Orlidge and Hollis, 1982). Briefly, the N-methylation of histamine was carried out in the presence of [methyf-3H]SAMe and histamine methyltransferase solution prepared from rat kidneys. Following incubation, samples were boiled and subjected to two extractions in toluene and isoamyl alcohol with intervening desiccation. Samples were counted on a Packard Tri-Carb scintillation counter. Counts in disintegrations per minute were regressed against known standards for the determination of histamine parameters. Histamine content was determined from samples incubated in D-histidine, and histidine decarboxylase activity, from samples incubated with L-histidine. Standard curves with and without 10e4 g/ml were compared to ensure that residual heparin from the cell lysate did not interfere with the N-methylation of histamine. Final histidine decarboxylase activity was expressed as nanograms of histamine formed per 100,000 cells per hour and histamine content as nanograms per 100,000 cells. Data were analyzed by one factor analysis of variance. Significance by this test was followed by appropriate tests for multiple comparisons. Group means with equal sample number were compared using Duncan’s multiple range test. The Newman-Keuls test for multiple comparison was used to compare means from groups unequal in sample number. RESULTS Untreated endothelial cell cultures exhibit a density-related decline in histamine content (Fig. 1). This fall in cellular histamine content amounts to a 94% decline between cultures plated at 16,000 cells/cm2 and those plated at 1000 cells/ cm2. Histidine decarboxylase activity, on the other hand, declines insignificantly with an increase in the plating density of 3-day cultures (Fig. 1). Figure 2 depicts the effects of 10e7 g/ml heparin on histamine content in endothelial cell cultures over the same range of plating densities. The effects of exogenous heparin on this parameter is significant only at the low plating density, where a 67% contraction of the nascent histamine pool is observed. While effecting a drop in endothelial histamine content, heparin treatment resulted in a density-dependent and dose-related stimulation of histidine decarboxylase (HD) activity (Fig. 3). The overall level of HD activity and the degree of
96
BLANK
:t T
AND HOLLIS
\
Ix IO’ PLATING
4x10’ DENSITY (cells/cm’
I I
,6x10’
)
FIG. 2. Effects of heparin (IO-’ g/ml) on histamine content in BAEC cultures plated at varying
stimulation by heparin treatment was greatest in subconfluent cultures of endothelium and progressively less as plating density was increased. Somewhat surprisingly, the activity of HD does not parallel the heparin-induced contraction of the histamine pool (Fig. 4). While histamine content falls about 67% with heparin treatment, HD activity is stimulated in a dose-related manner up to almost fivefold at the highest concentration tested. Heparin at 10T7 g/ml was as effective as higher concentrations in causing the decrease in histamine content. Untreated aortic SMC also exhibit a density-related decline in histamine content (Fig. 5). However, the response of the SMC histamine pool to heparin treatment is decidedly different from that of the bovine aortic endothelial cell (BAEC) (Fig. 6). First, the absolute level of histamine in control BAEC cultures is more than twice that of control SMC. Second, BAEC responds to heparin treatment by a 67% contraction of the nascent histamine pool. SMC do not exhibit any significant change in this parameter over the range of heparin concentrations tested. However, in low density SMC cultures, heparin in concentrations between lop7 and 1O-4 g/ml also stimulated HD activity in a dose-related manner. At 1O-4
FIG, 3. Dose-response
curve depicting the effects of heparin on HD activity at varying plating
EFFECTS
OF HEPARIN
0
ON ENDOTHELIAL
16’ HEPARIN
16” DOSAGE
CELL
HISTAMINE
Id’
97
Id’
(g /ml)
FIG. 4. Comparison of the dose-response curves for both HD activity and histamine content with heparin treatment in low density BAEC cultures.
g/ml this represented a fivefold increase over control values. Again, activity of HD does not correlate well with the histamine curve, suggesting the significance of compensating metabolic processes (Fig. 7). In further experiments, the effects of protamine sulfate on heparin-induced alterations in histamine content were examined. Again, treatment with heparin caused a greater than 50% drop in cellular histamine content (Fig. 8). Protamine sulfate (lO-‘j g/ml) completely reversed the effects of heparin on this parameter, while protamine sulfate alone did not alter histamine content significantly from the control or from either of the treatments. This effect of heparin is not only inhibitable, but it is relatively specific in that chondroitin sulfate and hyaluronic acid failed to effect a significant contraction of the BAEC nascent histamine pool (Fig. 9). Finally, 72-hr culture densities for BAEC and SMC cultures are reported in Tables 1 and 2, respectively. These data demonstrate that final cell densities parallel the initial plating density and suggest that neither BAEC nor SMC cultures plated at 1000 cells/cm2 are at a proliferative disadvantage relative to those plated
‘t
i’
\
1-d IX IO'
PLATING
4x IO’ DENSITY (cells/cm*
I6.10* )
FIG. 5. Histamine content and HD activity as a function of plating density in 3-day cultures of SMC.
BLANK
AND HOLLIS
.. 6 ..5
I 10.’
0
HEPARIN
FIG. 6. Comparison of the dose-response cultures with heparin treatment.
IODOSAGE
1 g/ml
10.’ 1
16’
curves for histamine content between BAEC and SMC
at higher densities, though some of the reagents tested, heparin and hyaluronic acid, have dramatic effects on final cell density. DISCUSSION It is generally agreed that the vascular endothelium serves as a permeability barrier against lumenal materials entering the arterial wall. Most of the mammalian arterial wall is avascular and depends on bloodborne materials for nutrition. The endothelium must then regulate the type and amount of molecules passing through the intimal layer. Furthermore, it is believed that disruption of the endothelial barrier, though perhaps not obligatory, nevertheless contributes to intimal thickening via the subsequent interaction of growth factors with underlying smooth muscle cells (Ross, 1986). Disruption of the endothelial barrier occurs with all types of endothelial injury and is accompanied by increases in vessel wall permeability. Aortic histamine
I 0
I Id’
lo-” HEPPiRlN
DOSAGE
16’
16’
(g/ml)
FIG. 7. Effects of varying doses of heparin on histamine content and HD activity in low density SMC cultures.
EFFECTS OF HEPARIN
ON ENDOTHELIAL
FIG. 8. Effects of protamine sulfate on the heparin-induced density BAEC cultures.
CELL HISTAMINE
99
contraction of the histamine pool in low
metabolism is altered in a variety of atherogenic risk situations, including diabetes (Orlidge and Hollis, 1982; Gallik and Hollis, 1981), hypercholesterolemia (Markle and Hollis, 1975; Hollis and Sloss, 1975), and shear stress (DeForrest and Hollis, 1980; Paraschos and Hollis, 1985). Orlidge and Hollis (1982) reported that in streptozotocin-diabetic rats, aortic endothelial cell histamine content increased 138%, HD activity increased 250%, and histaminase activity decreased 50% over the 4-week holding period. In the underlying smooth muscle the histamine content increased over 150% and the HD activity increased over 300% with respect to control values. Insulin treatment completely reversed these metabolic alterations. Other work has also indicated that the histamine-forming capacity (HFC) of the aorta is increased in cholesterol-fed rabbits and that this increased HFC is intimately associated with an increased uptake into the arterial wall (Owens and Hollis, 1979; Hollis and Furniss, 1980). Owens and Hollis (1979) demonstrated that the partial inhibition of HD activity in New Zealand White rabbits resulted in a 51% reduction in aortic albumin up-
FIG. 9. The effects of chondroitin cultures plated at low density.
sulfate and hyaluronic acid on the histamine content of BAEC
100
BLANK
AND HOLLIS
TABLE 1 Final BAEC Densities in Cultures Plated at 1000. 4000, and 16,000 Cells/cm*
Treatment Control Heparin
Chondroitin sulfate Hyaluronic acid Protamine sulfate Protamine sulfate + heparin
Concentration kidml) IO-’ IO-6 10-S 10-4 10-4 10-4 IO-6 10-6 10-d
Initial plating density (cells/cm*) 1000
4000
16,000
1.14 0.97 0.70 0.71 1.00 1.09 0.65 1.27 0.89
3.73 4.32 3.60 3.57 3.68 -
8.85 8.78 9.12 8.68 9.42 -
Note. Only cultures plated at the lowest density were treated with chondroitin sulfate (10m4 g/ml), hyaluronic acid ( 1O-4 g/ml), and protamine sulfate ( 10e6 g/ml) for comparison to the effects of heparin. Higher density cultures were unresponsive to heparin treatment.
take and a 63% reduction in oil red 0 staining in thoracic aortas from rabbits fed a cholesterol diet. Later, Owens and Hollis (1981), using Evans blue dye to identify areas of spontaneous arterial injury in canine aortas, found a significant correlation between local albumin accumulation, HD activity, and histamine content. There are other reports as well of an expansion of this nascent histamine pool in hypertension (Bolitho and Hollis, 1975). Data from this laboratory have also confirmed an increase in the histamine content of cultured BAEC subjected to elevated shear stress (Paraschos and Hollis, 1985). Finally, Kalsner and Richards (1984) have found marked increases in the histamine content of coronary arteries from hearts of cardiac patients and in vessel segments assessed as atherosclerotic. While the relationship between the histamine content of various tissues and conditions of injury are well established, specific effects of this nascent histamine pool expansion are less well understood. Both BAEC and SMC exhibit a dramatic density-dependent decline in histamine content. Higher density cultures of both cell types are relatively depleted of this amine. In view of the data presented here, it appears that the size of the nascent pool may be related directly or indirectly to the rate of cell division and turnover. Indeed, exogenous histamine has been shown to stimulate the growth of nonquiescent BAEC (D’Amore and Shepro, 1977). Experimental conditions such as hypercholesterolemia which induce an expansion of the aortic histamine pool (Hollis and Furniss, 1980; Owens and Hollis, 1979) also predispose the endothelium to injury or possible desquamation (Massman and Jellinek, 1980). In either case, the increase in cell turnover under these conditions may be associated with an acute expansion of the histamine pool and resultant alterations in transmural permeability. The effects of histamine on decreasing actin cables in endothelium may, in part, explain the action of this amine in inflammatory permeability (Welles et al., 1985). It is of interest that in this study contraction of the BAEC histamine pool was observed only in cultures plated at low density. At a confluent density, endothelial cells secrete heparin-like glycosaminoglycans which affect SMC and BAEC metabolism (Fritze et al., 1985; Gadjusek, 1985). That the density-related decline
EFFECTS OF HEPARIN
ON ENDOTHELIAL
TABLE 11 Final SMC Densities in Cultures Treated with Heparin (lo-’
Control Heparin
to 10e4 g/ml)
Initial plating density (cells/cm*)
Concentration (g/ml)
Treatment
101
CELL HISTAMINE
10-7 IO-6 10-s IO-4
1000
4000
16.000
1.43 1.38 0.85 0.75 0.57
5.38 4.98 4.71 3.50 I .93
13.00 11.63 11.40 9.90 7.95
in BAEC histamine content may be associated with a concomitant rise in endogenous heparins is suggested by the failure of exogenous heparin to affect this parameter in cultures plated at higher densities. The control point of the putative regulation has not been elucidated in this study. However, it appears that heparin may regulate the overall level of the histamine pool by modulating histamine catabolism. This is suggested indirectly by several lines of evidence. First, heparin stimulates activity of the synthetic enzyme in both SMC and BAEC in a dose-related manner, while effecting a histamine pool contraction only in BAEC cultures. Second, the density-related decline in histamine content in untreated cultures of BAEC and SMC appears to be independent of changes in the activity of HD. Finally, heparin has been shown to enhance catabolic pathways for histamine in other systems (Hahn et al., 1966). ACKNOWLEDGMENTS The authors thank Robert Sapanski, Charles Smith, and Mark Schmidt for their invaluable technical assistance and Dr. Patricia D’Amore and Dr. Alicia Orlidge for their generous gift of smooth muscle cells. We are also indebted to Mary Alice Shea for the typing of this manuscript.
REFERENCES BALUDA. V. I?, SUSHKEVICH. G. N., and LIASKO, L. I. (1982). Effect of heparin on prostacychn activity. Biul. Eksp. Biol. Med. 94, 79-80. BERENSON, G. S.. RADHAKRISHNAMURTHY. B.. SRINIVASAN, S. R., VIJAYAGOPAL, P., and DALFERES. E. R. (1985). Proteoglycans and potential mechanisms related to atherosclerosis. Ann. N. Y. Acad.
Sci. 454, 69-78.
BOLITHO. G. A., and HOLLIS, T. M. (1975). Aortic histamine synthesis in experimental hypertension. Proc.
Sot.
Exp.
Biol.
Med.
148, I I89-
I 192.
CASTELLOT, J. J., JR.. ADDONIZIO. M. L., ROSENBERG. R., and KARNOVSKY, M. J. (1981). Cultured endothelial cells produce a heparin-like inhibitor of smooth muscle cell growth. J. Cell Biol. 90, 372-379. CLOWES. A. W., and CLOWES. M. M. (1984). Heparin inhibits injury-induced arterial smooth muscle migration and proliferation. Fed. Proc. 43, 459. CLOWES, A. W.. and KARNOVSKY, M. J. (1977). Suppression by heparin of smooth muscle cell proliferation in injured arteries. Nrrtlrre (London) 265, 625-626. D’AMORE, P., and SHEPRO, D. (1977). Stimulation of growth and calcium influx in cultured bovine aortic endothelial cells by platelets and vasoactive substances. J. Cell Physiol. 92, 177- 184. DEFOREST, J. M., and HOLLIS. T. M. (1980). Relationship between low intensity shear stress, aortic histamine formation, and aortic albumin uptake. Exp. Mol. Putho/. 32, 217-225. ENGLEBERG, H. (1977). Probable physiological functions of heparin. Fed. Proc. 36, 70-72. FRITZE, L. M. S., REILLY, C. F., and ROSENBERG, R. (1985). An antiproliferative heparin sulfate species produced by postconfluent smooth muscle cells. J. Cell Biol. 100, 1041- 1049.
102
BLANK
AND
HOLLIS
GADJUSEK, C. M. (1985). Heparin release of endothelial cell-derived mitogens. Fed. Proc. 44, 1655. GALLIK, S., and HOLLIS, T. M. (1981). Studies on aortic histamine synthesis in experimental diabetes. Proc. Sot. Exp. Biol. Med. 166, 496-500. GLIMELIUS, B., BUSCH, C., and HOOK, M. (1978). Binding of heparin on surface of cultured human endothelial cells. Thromb. Res. 12, 773-782. GUYTON, J. R., ROSENBERG, R. D., CLOWES, A. W., and KARNOVSKY, M. J. (1980). Inhibition of rat arterial smooth muscle cell proliferation by heparin. Circ. Res. 46, 625-634. HAHN, F., SCHMUTZLER, W., SESEKE, G., GIERTZ, H., and BERNAUER. W. (1966). Histaminasefreisetzung durch heparin und protamin beim meerschweinchen. Biochem. Pharmacol. 15, 155-160. HIEBERT, L. M., and JAQUES, L. B. (1976). Heparin uptake on endothelium. Artery 2, 26-37. HOLLIS. T. M., and FURNISS, J. V. (1980). Relationship between aortic histamine formation and aortic albumin permeability in atherogenesis. Proc. Sot. Exp. Biol. Med. 165, 271-274. HOLLIS, T. M., and SLOSS, R. J. (1975). Rabbit aortic histamine synthesis following short term cholesterol feeding. Atherosclerosis 21, 125-134. JAQUES, L. B., and WATERS, L. B. (1941). The identity and origin of the anti-coagulant of anaphylactic shock in the dog. .I. Physiol. 99, 454-466. KALSNER, J., and RICHARDS, R. (1984). Coronary arteries of cardiac patients are hyperreactive and contain stores of amines: A mechanism for coronary spasm. Science 223, 143.5-1437. MARKLE, R. A., and HOLLIS. T. M. (1975). Rabbit aortic endothelial and medial histamine synthesis following short-term cholesterol feeding. Esp. Mol. Puthol. 23, 417-426. MASSMAN, J.. and JELLINEK, H. (1980). Hematogenic infiltration of the aortic intima in normal and hypercholesterolemic swine: Studies on en face endothelium-intima preparations. Exp. Mol. Parhol.
18, 1 I-24.
ORLIDGE. A., and HOLLIS, T. M. (1982). Aortic endothelial and smooth muscle histamine metabolism in experimental diabetes. Arferiosclereosis 21, 125- 134. OWENS, G. K., and HOLLIS, T. M. (1979). Relationship between inhibition of aortic histamine formation, aortic albumin permeability and atherosclerosis. Atherosclerosis 34, 365-373. OWENS, G. K., and HOLLIS, T. M. (1981). Local aortic histamine metabolism and albumin accumulation: differences between blue and white areas. Arrheriosclerosis 1, 265-272. PARASCHOS, S. I., and HOLLIS, T. M. (1985). Effects of shear stress on bovine endothelial cells, in vitro, with relation to the pathogenesis of atherosclerosis. Fed. Proc. 44, 1659. PEARSON, J. D. (1983). Endothelial cell metabolism. Prog. Appl. Microcirc. 1, Il4- 126. ROSS, R. (1986). The pathogenesis of atherosclerosis-an update. N. Engl. J. Med. 314, 488-500. SCHWARTZ, S., GADJUSEK, C., REIDY, M.. SELDEN, S., III, and HAUDENSCHILD. C. (1980). Maintenance of integrity in aortic endothelium. Fed. Proc. 39, 2618-2624. TAYLOR, K. M., and SNYDER, S. H. (1972). Dynamics of the regulation of histamine levels in mouse brain. J. Neurochem. 19, 341-354. WELLES, S. L., SHEPRO, D., and HECHTMAN, H. B. (1985). Vasoactive amines modulate actin cables (stress fibers) and surface area in cultured bovine endothelium. J. Cell. Physiol. 123, 337-342. WILLIAMS, M. P., STREETER, H. B., WUSTEMAN. F. S., and CRYER, A. (1983). Heparan sulphate and the binding of lipoprotein lipase to porcine thoracic aorta endothelium. Biochim. Biophys. Acta 761, 83-91.