European Journal of clinical Investigation (1979) 9 , 5-10
The effects of albumin bound fatty acids on the platelet inhibitory function of human endothelial cells ARNE NORDOY, BIRGIT SVENSSON & JOHN C. HOAK,Departments of Medicine, University of Iowa Hospitals, Iowa City, Iowa, U.S.A., and University of Tromsb, Tromsb, Norway Received 26 June 1978 and in revised form 5 September 1978
Abstract. This study was carried out to evaluate the effects of albumin-bound fatty acids on the anti-platelet effects of endothelial cells. Primary cultures of human endothelial cells (ECM), grown in confluent monolayers, were incubated with plasma or growth medium enriched with albumin-bound fatty acids (FA) for 2-20 h. The effects of ECM on ADP-induced platelet aggregation (PA) and collagen-induced PA and prostaglandin synthesis in platelet-rich plasma were tested. ECM released an inhibitor of platelet reactions which resembled the activity of PGIz (prostacyclin). The inhibitory activity was increased by preincubation of ECM with arachidonic acid (AA). A moderate decrease of the activity was obtained by incubation with longchain saturated, monoenoic and dienoic unsaturated fatty acids. A pronounced decrease of the inhibitor was obtained by incubation with di-homo-y-linolenic acid (DHLA). Paired combinations of AA with the other fatty acids in the incubation medium partially restored the inhibitor activity obtained by the separate FA. The stimulation of the inhibitor by AA was dose dependent and high concentrations of AA reduced this activity. The present study indicates that the quantity and quality of the plasma free fatty acids can affect the endothelial cells’ ability to act as a non-thrombogenic surface.
Key words. Fatty acids, endothelial cells, platelets, prostaglandins.
acids may have the opposite effect [for review see 31. However, arachidonic acid (AA) may directly influence thrombus development by the formation of thromboxane A2, a potent platelet aggregating substance generated in platelets [4], or by production of PG12 (prostacyclin), a potent inhibitor of platelet function, formed in endothelial cells [5-71. Di-homo-y-linolenic acid (DHLA), a precursor of the monoenoic prostaglandins, may inhibit platelet aggregation by formation of PGEl in the platelets [8]. This acid has been suggested for use as an antithrombotic drug 19, 101. Prolonged exposure of platelets, endothelial cells and other cell types t o various fatty acids, either by dietary manipulations or by cell culture studies [12-181, may induce profound changes in the phospholipid composition of the cell membranes. In the present study using in vitro cultures, we have exposed human endothelial cells to various fatty acids, bound t o albumin, and then examined the inhibitory effect of the endothelial cells upon platelet function. Materials and Methods
Endothelial cell cultures. Primary cultures of end@ thelial cells were prepared from human umbilical veins according t o the method of Jaffe et al. [19] with a slight modification as recently described [20]. Confluent endothelial cell monolayers (ECM) were used for further studies 4 days after seeding on plastic petri dishes (25 X 10 mm) (Falcon Plastics, Oxnard, Ca.). Empty dishes (ED) washed with the culture medium were used as controls.
Introduction
High plasma concentrations of free fatty acids have been associated with an increased tendency t o thrombosis in humans and in experimental animals [ l , 21. In vitro studies have shown that long-chain saturated fatty acids may induce platelet aggregation and increase platelet adhesiveness, whereas longchain polyunsaturated fatty Correspondence: Dr Arne Nor day, Department of Medicine, University Hospital, 9012 Tromsq4, Norway. 0014-2972/79/0200-0005 $02.00 0 1979 Blackwell Scientific Publications
Preparation on the albumin-FFA solution. Fatty acids (99% pure) were purchased from Nu Chek Prep. Inc. (Elysian, MN) with the exception of di-homo-ylinolenic acid which was kindly given t o us by Dr K. F. Stone, Roche Products Ltd, Hertfordshire, England. The following fatty acids were used: stearic acid (18:O); oleic acid (18:l); linolenic acid (9,12-18:2); di-homo-ylinolenic acid (8,11,14-20:3) and arachidonic acid (5,8,11,14-20:4). Human albumin, Fr. V, fatty acid-free was obtained from Miles Research Products (Miles
5
6
A. NORDQY, B. SVENSSON & J. C. HOAK
Laboratories, Inc., Elkhart, IN). The FFA-albumin solution was prepared as earlier described [21] using Celite (Johns-ManviUe, Products Corp., Celite Div., Denver, CO) as the initial absorbent for FA. A 0.5 pmollml albumin solution with 0.140 mol/l NaC1, 0.0058 mol/l KC1 and 0.016 mol/l Tris buffer (Trizma Base, Sigma Chemical Co., St Louis, MO) with a final pH adjusted to 7.4 with 1 N HCI, and a titratable acidity [22] of 0.17 pmol of FFA per ml was used. The resultant FFAalbumin solution was extracted and analysed for FFA concentration [22]. FA:albumin ratios of 6.8 for stearic acid increasing to 18.6 for arachldonic acid were obtained. The FA-albumin solution was diluted with albumin to the desired concentration. The FA-albumin solutions were kept in sealed glass tubes under nitrogen at 4°C and were used within 2 weeks of preparation. At the end of this period, aliquots of the solutions were extracted [22], methylated for 10 min at 95°C with 14% BF3-CH30H and examined by glass liquid chromatography using a Hewlett-Packard 5710A GLC having a 6 ft by & in. metal column packed with Supelco Sp 2340. Peak areas were quantitated with an HP Integrator 3380 A using an internal standard, and peaks were identified by comparison of retention times to those of standards obtained from Applied Science Laboratories. At the lowest attenuation, only single peaks were observed in all solutions and the recovery was from 88% t o 99%. Platelet studies. Venous blood was collected from healthy, fasting donors who had not taken any drugs for the previous 14 days. Nine volumes of blood were collected in one volume of 3.2% trisodium citrate dihydrate and platelet-rich (PRP) and platelet-poor (PPP) plasma were prepared by centrifugation at 20°C at 270 g for 15 min and 1200 g for 30 min respectively. The platelet number in PRP was adjusted to 300,OOO/pl by addition of PPP. PRP was kept at 22°C and was used withln 4 h after sampling. Platelets were counted in a Coulter Counter (Model B, Coulter Electronics, inc., Hialeah, FL). Platelet aggregation was recorded according t o the method of Born 1231 using a Payton Aggregometer module (Payton Ass., Buffalo, NY) at 37°C. The percentage of aggregation registered at 6 min after addition of the aggregating agent was used as the endpoint of aggregation, using the light transmission in PPP as 100% and PRP without addition of the aggregating agent as 0%. ADP, 6.6 pmol/l final concentration in Tris buffered saline (TBS) (139 mmol/l NaCl and 15 mmol/l Tris, pH 7.5) (Sigma Chemical Corp., St Louis, MO), and collagen 50 pg/ml, dissolved in diluted acetic acid [14] (Sigma) were used as aggregating agents. Malondialdehyde (MDA) was measured in PRP samples from the aggregometer 6 min after the addition of collagen according to the method of Smith et al. [25]. Test procedure. This method has been detailed elsewhere 1241 and was performed as follows: 4 days after the dishes were seeded with cells, the medium was aspirated from the dishes, and the cells were washed three
times with 1 ml modified Hank's Balanced Salt Solution (MHBSS) [24]. They were then incubated with 1.5 ml of FA-albumin solution diluted in PPP to the desired concentration for the desired length of time at 37°C in a metabolic incubator. For the initial 15 min of the incubation period the dishes were placed on a rocker platform with a 30" tilt (Bellco Glass Inc., Vineland, NS) and rocked continuously at a rate of 10 cycles per minute. At the end of the incubation period the FA solution was pipetted off and the dishes were washed three times with 1 ml MHBSS. They were then incubated with 1.5 ml Tris buffer (0.05 mol/l, pH 7.5) for another 5 min on the rocker platform. 0.3 ml of this buffer was then added to 0.45 PRP in the aggregometer cuvettes and ADP-induced platelet aggregation was recorded. In order to test the direct effect of ECM on platelets, the rest of the buffer solution was aspirated from the dishes and replaced with 1.5 ml PRP for an additonal incubation period of 30 min on the rocker platform at 37°C. 0.9 ml of this PRP was collected and collagen-induced platelet aggregation and MDA production were determined. in additional experiments the endothelial cells were incubated for 15 min with indomethacin (Merck-Sharp 13Dohme) (1 mglml) in TBS at 37°C before they were incubated with albumin bound AA and DHLA in PRP. in other experiments the endothelial cell monolayers were incubated with a modified culture medium (the FA-albumin solution replaced the 20% fetal calf serum for the last 20 h of the growth period). The dishes were washed three times with 1 ml MHBSS at the end of this period and the effects of the ECM on platelet function were performed as described above. The endothelial cell monolayers were always inspected with phase microscopy before use t o ensure the presence of normal, confluent monolayers of cells. Statistics. Student's 't' test for small samples was used. Results
The endothelial cells showed slight granulation when they had been incubated with arachidonic acid or di-homo?linolenic acid bound to albumin for 20 h. Otherwise the cell cultures looked healthy without significant changes compared with cells grown in the standard-culture medium. Effect of short-term incubation ( 2 h) ofECM with plasma enriched with albuminlFA
Samples of 1.2 ml PPP and 0.3 ml FA-albumin solution (ratio 4.5 and 9) were mixed and incubated with ECM for 2 h. When the FA-albumin solutions with ratios of FA to albumin of 4.5 and 9 were added to PPP, the content of the actual FA in the plasma samples was increased by 0.45 and 0.90 pmol/ml PPP respectively. As shown in Table 1, the inhibitor of ADP- and collagen-induced platelet aggregation and MDA produc-
THE EFFECTS OF FATTY ACIDS ON ENDOTHELIUM
7
Table 1. The effect of endothelial cell monolayers preincubated for 2 h with fatty acid bound albumin
diluted in platelet poor plasma on ADP-induced platelet aggregation (PA) and collagen-induced PA and malondialdehyde (MDA formation in platelet rich plasma). Controls incubated with 20%albumin solution in plasma with and without (ED) endothelial cells. Mean and SEM of six experiments.
ADP-PA (%) Albuminbound FA
Collagen-MDA (nglml)
Collagen-PA (%)
4.5*
9.0*
4.5*
9.0*
4.5*
1 2 f 2$ 13 f 3$ 12r6 6 i 2 1+1 14 + 5 $ 68f55
l o + 3$ 1 0 f 3$
54f 11 55 f 7
13 i 3$ 1 8 + 15 1 i 1 14 + 2$ 68f 55
55 f 8 5 5 f 11 4 1 + 12 53 * 8 80f3d
5 5 f 10 5 9 + 11 58 + 9$ 61+9$ 4 0 t 13 56 f 8 80+3p
44 f 42f 45 + 54f 31 t 5Of 78 +
9.0*
~~
18:Ot 18:l 18:2 20:3 20:4 Albumin AlbuminED
14 11 14 19 20 17 205
48r 45 t 58 f 64 f 38 +
18 18 19$ 17$ 19 S O * 17 78 f 205
* Ratio FA/Albumin. Ratio of C I8:O-albumin solution used was not 9, but was 6.8. In order to keep the final concentration of C 18:O comparable to the studies with the other fatty acids, 0.39 ml of C 18:O-alb was added in place of 0.3 ml. $ Significance of difference compared with 20:4;P < 0.05. 0 Significance of difference compared with 20 :4 ;P < 0.0 1.
tion released from the endothelial cells was released from all cell cultures with both concentrations of FA and also when only FA-poor albumin was added t o the incubation mixture. The inhibitory effect was greater after incubation with arachidonic acid (20:4/alb) than with any of the other fatty acids. However, no greater effect was observed when the ratio of 20:4 t o albumin was increased from 4.5 t o 9.0, suggesting that a maximal stimulation of inhibitor production and/or release had been achieved with the lower concentration. Compared with the effect of arachidonic acid, none of the other fatty acids tested stimulated endothelial cells' ability to counteract platelet aggregation and MDA production. In fact, when the highest concentration was used, DHLA seemed t o reduce the formation and/or release of the inhibitor below the level obtained by only FA-poor albumin. These results may indicate that the two polyunsaturated fatty acids AA and DHLA directly
Sian of difference lo 20 4 p
I
801
* * S i i n of difference lo 20 4 p
..
The effects of albumin bound fatty acids on the platelet inhibitory function of human endothelial cells ARNE NORDOY, BIRGIT SVENSSON & JOHN C. HOAK,Departments of Medicine, University of Iowa Hospitals, Iowa City, Iowa, U.S.A., and University of Tromsb, Tromsb, Norway Received 26 June 1978 and in revised form 5 September 1978
Abstract. This study was carried out to evaluate the effects of albumin-bound fatty acids on the anti-platelet effects of endothelial cells. Primary cultures of human endothelial cells (ECM), grown in confluent monolayers, were incubated with plasma or growth medium enriched with albumin-bound fatty acids (FA) for 2-20 h. The effects of ECM on ADP-induced platelet aggregation (PA) and collagen-induced PA and prostaglandin synthesis in platelet-rich plasma were tested. ECM released an inhibitor of platelet reactions which resembled the activity of PGIz (prostacyclin). The inhibitory activity was increased by preincubation of ECM with arachidonic acid (AA). A moderate decrease of the activity was obtained by incubation with longchain saturated, monoenoic and dienoic unsaturated fatty acids. A pronounced decrease of the inhibitor was obtained by incubation with di-homo-y-linolenic acid (DHLA). Paired combinations of AA with the other fatty acids in the incubation medium partially restored the inhibitor activity obtained by the separate FA. The stimulation of the inhibitor by AA was dose dependent and high concentrations of AA reduced this activity. The present study indicates that the quantity and quality of the plasma free fatty acids can affect the endothelial cells’ ability to act as a non-thrombogenic surface.
Key words. Fatty acids, endothelial cells, platelets, prostaglandins.
acids may have the opposite effect [for review see 31. However, arachidonic acid (AA) may directly influence thrombus development by the formation of thromboxane A2, a potent platelet aggregating substance generated in platelets [4], or by production of PG12 (prostacyclin), a potent inhibitor of platelet function, formed in endothelial cells [5-71. Di-homo-y-linolenic acid (DHLA), a precursor of the monoenoic prostaglandins, may inhibit platelet aggregation by formation of PGEl in the platelets [8]. This acid has been suggested for use as an antithrombotic drug 19, 101. Prolonged exposure of platelets, endothelial cells and other cell types t o various fatty acids, either by dietary manipulations or by cell culture studies [12-181, may induce profound changes in the phospholipid composition of the cell membranes. In the present study using in vitro cultures, we have exposed human endothelial cells to various fatty acids, bound t o albumin, and then examined the inhibitory effect of the endothelial cells upon platelet function. Materials and Methods
Endothelial cell cultures. Primary cultures of end@ thelial cells were prepared from human umbilical veins according t o the method of Jaffe et al. [19] with a slight modification as recently described [20]. Confluent endothelial cell monolayers (ECM) were used for further studies 4 days after seeding on plastic petri dishes (25 X 10 mm) (Falcon Plastics, Oxnard, Ca.). Empty dishes (ED) washed with the culture medium were used as controls.
Introduction
High plasma concentrations of free fatty acids have been associated with an increased tendency t o thrombosis in humans and in experimental animals [ l , 21. In vitro studies have shown that long-chain saturated fatty acids may induce platelet aggregation and increase platelet adhesiveness, whereas longchain polyunsaturated fatty Correspondence: Dr Arne Nor day, Department of Medicine, University Hospital, 9012 Tromsq4, Norway. 0014-2972/79/0200-0005 $02.00 0 1979 Blackwell Scientific Publications
Preparation on the albumin-FFA solution. Fatty acids (99% pure) were purchased from Nu Chek Prep. Inc. (Elysian, MN) with the exception of di-homo-ylinolenic acid which was kindly given t o us by Dr K. F. Stone, Roche Products Ltd, Hertfordshire, England. The following fatty acids were used: stearic acid (18:O); oleic acid (18:l); linolenic acid (9,12-18:2); di-homo-ylinolenic acid (8,11,14-20:3) and arachidonic acid (5,8,11,14-20:4). Human albumin, Fr. V, fatty acid-free was obtained from Miles Research Products (Miles
5
6
A. NORDQY, B. SVENSSON & J. C. HOAK
Laboratories, Inc., Elkhart, IN). The FFA-albumin solution was prepared as earlier described [21] using Celite (Johns-ManviUe, Products Corp., Celite Div., Denver, CO) as the initial absorbent for FA. A 0.5 pmollml albumin solution with 0.140 mol/l NaC1, 0.0058 mol/l KC1 and 0.016 mol/l Tris buffer (Trizma Base, Sigma Chemical Co., St Louis, MO) with a final pH adjusted to 7.4 with 1 N HCI, and a titratable acidity [22] of 0.17 pmol of FFA per ml was used. The resultant FFAalbumin solution was extracted and analysed for FFA concentration [22]. FA:albumin ratios of 6.8 for stearic acid increasing to 18.6 for arachldonic acid were obtained. The FA-albumin solution was diluted with albumin to the desired concentration. The FA-albumin solutions were kept in sealed glass tubes under nitrogen at 4°C and were used within 2 weeks of preparation. At the end of this period, aliquots of the solutions were extracted [22], methylated for 10 min at 95°C with 14% BF3-CH30H and examined by glass liquid chromatography using a Hewlett-Packard 5710A GLC having a 6 ft by & in. metal column packed with Supelco Sp 2340. Peak areas were quantitated with an HP Integrator 3380 A using an internal standard, and peaks were identified by comparison of retention times to those of standards obtained from Applied Science Laboratories. At the lowest attenuation, only single peaks were observed in all solutions and the recovery was from 88% t o 99%. Platelet studies. Venous blood was collected from healthy, fasting donors who had not taken any drugs for the previous 14 days. Nine volumes of blood were collected in one volume of 3.2% trisodium citrate dihydrate and platelet-rich (PRP) and platelet-poor (PPP) plasma were prepared by centrifugation at 20°C at 270 g for 15 min and 1200 g for 30 min respectively. The platelet number in PRP was adjusted to 300,OOO/pl by addition of PPP. PRP was kept at 22°C and was used withln 4 h after sampling. Platelets were counted in a Coulter Counter (Model B, Coulter Electronics, inc., Hialeah, FL). Platelet aggregation was recorded according t o the method of Born 1231 using a Payton Aggregometer module (Payton Ass., Buffalo, NY) at 37°C. The percentage of aggregation registered at 6 min after addition of the aggregating agent was used as the endpoint of aggregation, using the light transmission in PPP as 100% and PRP without addition of the aggregating agent as 0%. ADP, 6.6 pmol/l final concentration in Tris buffered saline (TBS) (139 mmol/l NaCl and 15 mmol/l Tris, pH 7.5) (Sigma Chemical Corp., St Louis, MO), and collagen 50 pg/ml, dissolved in diluted acetic acid [14] (Sigma) were used as aggregating agents. Malondialdehyde (MDA) was measured in PRP samples from the aggregometer 6 min after the addition of collagen according to the method of Smith et al. [25]. Test procedure. This method has been detailed elsewhere 1241 and was performed as follows: 4 days after the dishes were seeded with cells, the medium was aspirated from the dishes, and the cells were washed three
times with 1 ml modified Hank's Balanced Salt Solution (MHBSS) [24]. They were then incubated with 1.5 ml of FA-albumin solution diluted in PPP to the desired concentration for the desired length of time at 37°C in a metabolic incubator. For the initial 15 min of the incubation period the dishes were placed on a rocker platform with a 30" tilt (Bellco Glass Inc., Vineland, NS) and rocked continuously at a rate of 10 cycles per minute. At the end of the incubation period the FA solution was pipetted off and the dishes were washed three times with 1 ml MHBSS. They were then incubated with 1.5 ml Tris buffer (0.05 mol/l, pH 7.5) for another 5 min on the rocker platform. 0.3 ml of this buffer was then added to 0.45 PRP in the aggregometer cuvettes and ADP-induced platelet aggregation was recorded. In order to test the direct effect of ECM on platelets, the rest of the buffer solution was aspirated from the dishes and replaced with 1.5 ml PRP for an additonal incubation period of 30 min on the rocker platform at 37°C. 0.9 ml of this PRP was collected and collagen-induced platelet aggregation and MDA production were determined. in additional experiments the endothelial cells were incubated for 15 min with indomethacin (Merck-Sharp 13Dohme) (1 mglml) in TBS at 37°C before they were incubated with albumin bound AA and DHLA in PRP. in other experiments the endothelial cell monolayers were incubated with a modified culture medium (the FA-albumin solution replaced the 20% fetal calf serum for the last 20 h of the growth period). The dishes were washed three times with 1 ml MHBSS at the end of this period and the effects of the ECM on platelet function were performed as described above. The endothelial cell monolayers were always inspected with phase microscopy before use t o ensure the presence of normal, confluent monolayers of cells. Statistics. Student's 't' test for small samples was used. Results
The endothelial cells showed slight granulation when they had been incubated with arachidonic acid or di-homo?linolenic acid bound to albumin for 20 h. Otherwise the cell cultures looked healthy without significant changes compared with cells grown in the standard-culture medium. Effect of short-term incubation ( 2 h) ofECM with plasma enriched with albuminlFA
Samples of 1.2 ml PPP and 0.3 ml FA-albumin solution (ratio 4.5 and 9) were mixed and incubated with ECM for 2 h. When the FA-albumin solutions with ratios of FA to albumin of 4.5 and 9 were added to PPP, the content of the actual FA in the plasma samples was increased by 0.45 and 0.90 pmol/ml PPP respectively. As shown in Table 1, the inhibitor of ADP- and collagen-induced platelet aggregation and MDA produc-
THE EFFECTS OF FATTY ACIDS ON ENDOTHELIUM
7
Table 1. The effect of endothelial cell monolayers preincubated for 2 h with fatty acid bound albumin
diluted in platelet poor plasma on ADP-induced platelet aggregation (PA) and collagen-induced PA and malondialdehyde (MDA formation in platelet rich plasma). Controls incubated with 20%albumin solution in plasma with and without (ED) endothelial cells. Mean and SEM of six experiments.
ADP-PA (%) Albuminbound FA
Collagen-MDA (nglml)
Collagen-PA (%)
4.5*
9.0*
4.5*
9.0*
4.5*
1 2 f 2$ 13 f 3$ 12r6 6 i 2 1+1 14 + 5 $ 68f55
l o + 3$ 1 0 f 3$
54f 11 55 f 7
13 i 3$ 1 8 + 15 1 i 1 14 + 2$ 68f 55
55 f 8 5 5 f 11 4 1 + 12 53 * 8 80f3d
5 5 f 10 5 9 + 11 58 + 9$ 61+9$ 4 0 t 13 56 f 8 80+3p
44 f 42f 45 + 54f 31 t 5Of 78 +
9.0*
~~
18:Ot 18:l 18:2 20:3 20:4 Albumin AlbuminED
14 11 14 19 20 17 205
48r 45 t 58 f 64 f 38 +
18 18 19$ 17$ 19 S O * 17 78 f 205
* Ratio FA/Albumin. Ratio of C I8:O-albumin solution used was not 9, but was 6.8. In order to keep the final concentration of C 18:O comparable to the studies with the other fatty acids, 0.39 ml of C 18:O-alb was added in place of 0.3 ml. $ Significance of difference compared with 20:4;P < 0.05. 0 Significance of difference compared with 20 :4 ;P < 0.0 1.
tion released from the endothelial cells was released from all cell cultures with both concentrations of FA and also when only FA-poor albumin was added t o the incubation mixture. The inhibitory effect was greater after incubation with arachidonic acid (20:4/alb) than with any of the other fatty acids. However, no greater effect was observed when the ratio of 20:4 t o albumin was increased from 4.5 t o 9.0, suggesting that a maximal stimulation of inhibitor production and/or release had been achieved with the lower concentration. Compared with the effect of arachidonic acid, none of the other fatty acids tested stimulated endothelial cells' ability to counteract platelet aggregation and MDA production. In fact, when the highest concentration was used, DHLA seemed t o reduce the formation and/or release of the inhibitor below the level obtained by only FA-poor albumin. These results may indicate that the two polyunsaturated fatty acids AA and DHLA directly
Sian of difference lo 20 4 p
I
801
* * S i i n of difference lo 20 4 p
..
