Reduced stimulatory activity on prostacyclin production by cultured endothelial cells in serum from aged and diabetic patients

Atherosclerosrs,

61

75 (1989) 61-66

Elsevier Scientific

Publishers

Ireland,

Ltd.

ATH 04246

Reduced stimulatory activity on prostacyclin production by cultured endothelial cells in serum from aged and diabetic patients F. Umeda, T. Inoguchi and H. Nawata Third Department

of Internal

Medicine,

Faculty

ofMedicine,

Kyurhu

Untuerstty,

Fukuoka

812 (Japan)

(Received 8 March, 1988) (Revised, received 27 July, 1988) (Accepted 29 August, 1988)

Reduced prostacyclin (PGI,) generation by the vascular wall shows a close relationship with the development of atherosclerosis. The present study found plasma-derived serum (PDS) to contain an activity which stimulated PGI, production by cultured bovine aortic endothelial cells. Diabetic and aged patients with atherosclerotic disease were examined for abnormalities in that stimulatory activity in PDS. PDS obtained from both diabetics (NIDDM) and aged patients showed a significant reduction in the stimulation of PGI, production by cultured bovine aortic endothelial cells compared with age-matched controls and young healthy volunteers, respectively. It was suggested that the reduced PGI, stimulatory activity in PDS may be one of the pathogenic mechanisms of vascular lesions such as atherosclerosis in diabetics and aged humans.

Key words: Atherosclerosis;

Prostacyclin;

Plasma-derived serum; NIDDM;

Introduction

Prostacyclin (PGI,) generated by the vascular wall acts as a potent vasodilator and an inhibitor of platelet adhesion and aggregation. PGI, works to prevent thrombus formation in vessels and the subsequent development of vascular lesions such as atherosclerosis. Recent studies have demonstrated that atherosclerotic vascular tissue gener-

Correspondence to: Dr. Fumio Umeda, Third Department of Internal Medicine, Faculty of Medicine, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka 812, Japan.

OO21-9150/89/$03.50

0 1989 Elsevier Scientific

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Ireland,

Aged humans

ates less PGI, [l] and that the circulating level of PGI, decreases with aging [2]. Reduced PGI, production by the vascular wall can be proposed as one possible pathogenic mechanism of atherosclerosis. MacIntyre et al. [3] found an activity in human plasma which stimulated PGI, production by cultured aortic endothelial cells. This stimulatory activity in plasma may be an important factor in the regulation of PGI, production by the vascular wall in vivo. Our previous studies [4,5] have also shown the presence of a PGI, stimulatory activity in human and rat plasma-derived serum (PDS). Furthermore, it was shown that the stimulatory Ltd

62 activity in PDS was reduced in human and experimental diabetics. The present study was undertaken to evaluate the abnormality in the PGI, stimulatory activity in PDS obtained from aged patients with atherosclerotic disease as well as from patients with non insulin-dependent diabetes mellitus.

metabolism or platelet function weeks prior to this study.

Preparation of plasma-derived serum (PDS) PDS was prepared according to Pledger’s method [6]. Briefly, after overnight fasting, blood was taken from an antecubital vein into a disposable syringe containing sodium citrate at a final concentration of 0.38%. Platelet poor plasma was immediately obtained by centrifugation at 1500 x g for 15 min, recalcified with 14 mM CaCl, and allowed to clot at 37 o C for 2 h. After recentrifugation, the supernatant was inactivated by heating at 56°C for 30 min. This specimen was used as PDS and was stored at -20’ C until the experiment.

Materials and Methods

Human subjects Male patients with non insulin-dependent diabetes mellitus (NIDDM, n = 11) and age- and sex-matched control subjects (n = 10) were selected for the present study. The glycemic control of diabetes in patients with NIDDM was indicated to be poor on the basis of fasting blood sugar (FBS) and HbA, levels. In addition, aged male patients with atherosclerotic disease such as ischemic heart disease and/or cerebral infarction (n = 10) and young healthy men (n = 10) were also selected for further study. The clinical features of the experimental human subjects are shown in Table 1. None of these had been taking any drugs which might affect prostaglandin

TABLE

for at least 2

Endothelial cell culture Endothelial cells were scraped from the thoracic aortic intima removed from l- to 2-year-old calves. As previously reported [4,5], the cells were cultured in Dulbecco’s modified Eagle’s medium (DME) supplemented with 10% fetal calf serum (FCS) and 100 pg/ml gentamicin at 37OC under an atmosphere of 95% air and 5% CO,. The medium was replaced twice weekly. Endothelial

1

CLINICAL

FINDINGS

IN THE SUBJECTS

OF THE STUDY

Results are the mean* SE. HbA,: hemoglobin A,; BP: blood infarction; IHD: ischemic heart disease; Ary: arrhythmia.

(1)

Duration

Age (y-s)

(years)

pressure;

Fasting blood sugar

Chol:

cholesterol;

Trig: triglyceride;

MI: myocardial

HbA,

Retinopathy

Proteinuria

(%)

(n)

(n)

(mg/dI) NIDDM

(n = 11)

43*4

Control (n = 10)

(2)

10*2

193*21

41+3

Age bears)

13.1+ 1.2

1:::

[_‘:I,” _

92k2 BP (mmHg)

Fasting blood sugar

Chol

Trig

(mg/dl)

(mg/dl)

ECG abnormality

Aortic calcification

Cerebral infarction

(mg/dl) Old (n = 10)

81&3

149*4/ 83f4

86&4

153*11

94+12

krs;;

Young (n = 10)

25*1

-

81k2

156f9

14+6

-

rf:;

_

[i46

63 cells were identified by phase-contrast microscopy as a typical monolayer growth and by measurement of von Willebrand’s factor using von Willebrand’s reagent purchased from Behringwerke AG (Marburg, F.R.G.). When confluent, the cells were passaged by trypsinization with 0.05% (w/v) trypsin solution. The cells from the 2nd to 7th passages were used in the present experiment. The trypsinized cells were placed in 24-well cluster dishes (Flow Laboratories Inc., McLean, VA) with 1 ml of DME containing 10% FCS. When the cells reached confluence (approx. 2 x lo4 cells/dish), the medium was changed to 1 ml of DME containing PDS specimens at various concentrations. After incubation for the time required, the medium was assayed for 6-keto-PGF,, (a stable breakdown product of PGI,).

mean f SE). The degradation of 6-keto-PGF,, in the medium was minimal over several hours at 37OC. The 6-keto-PGF,, concentration was measured by radioimmunoassay using a kit obtained from New England Nuclear Products (Boston, MA). The antibody against 6-keto-PGF,, was specific with less than 0.1% cross-reactivity with other prostaglandins. The bound and free ligands were separated using dextran-coated charcoal. The radioactivity in the supernatant was counted in an LSC-700 liquid scintillation counter (Aloka, Tokyo, Japan).

&Keto-PGF,, assay Extraction and purification of 6-keto-PGF,, from the medium was performed by a modification of the method of Jaffe et al. [7]. A l-ml aliquot of the medium was acidified with 0.1 N HCl and extracted twice with 5 ml ethyl acetate. The collected organic solvent was evaporated under N, gas and dissolved in absolute ethanol. The sample was kept at - 20°C until assay. On the day of assay, the ethanol solution was again evaporated and redissolved in 0.1 M phosphate buffer (pH 7.2) containing 1 M NaCl and 0.1% (w/v) gelatin. The overall recovery rate of 6-ketoPGF,, from the medium was 71.5 f 3.2% (n = 10,

Results

-

500

2 8

Statistical method Statistical analysis was carried out using Student’s t-test. All P values less than 0.05 were accepted as statistically significant.

6-Keto-PGF,, production by cultured bovine aortic endothelial cells was stimulated by the addition of human PDS pooled from 4 healthy volunteers in a time- and dose-dependent manner (Fig. 1). The maximal 6-keto-PGFia production stimulated by PDS was found at a final concentration of 10% in DME medium after incubation for 10 min, and that maximal production of 6-ketoPGF,, remained stable for 60 minutes. This time was, therefore, chosen for the measurement of 6-keto-PGF,, production by human PDS in all further experiments. Additionally, 6-keto-PGF,, 500

(A)

(B) T

A 0’

t

0

10 Time

20 ( min

30 1

1,

O

0

5

10

PDS concentration

15

20

(%I

Fig. 1. Stimulation of 6-keto-PGF,, production by cultured bovine aortic endothelial cells by pooled PDS from healthy volunteers (n = 4). (A) Time course following addition of 10% human PDS. (B) Dose response, cells were incubated for 60 min after addition of PDS. Results are shown as mean f SE of 4 experiments.

64 5oor

L

2 f=

u”

4 \ E

f

P < 0.001 ** 400

Control

24 ( n

10)

#JJ P
*

Diabetic

( n

11 )

L

A

o-

0

5

PDS concentration

z

Nlo-

g

m-

*u

.-s l5

Em L

F i &

loo-

0

I

I

0

5

10

1

10

PDS concentrationPM (5

)

Fig. 2. Comparison of 6-keto-PGF,, production after addition of PDS from diabetic patients and age-matched controls. Results are shown as mean+ SE. Significantly different, * P < 0.05, ** P -c0.001.

production stimulated by 10% PDS was enhanced approx. 3 times by the simultaneous addition of 50 PM exogenous arachidonic acid but inhibited completely after preincubation with 0.5 mM aspirin for 30 min. These results are consistent with the view that PDS prepared from human subjects contains a stimulatory activity on 6-ketoPGF,, production by cultured bovine aortic endothelial cells. 6-Keto-PGF,, production stimulated by PDS was examined in patients with NIDDM. As shown in Fig. 2, PDS obtained from patients with NIDDM showed a significant reduction in the stimulation of 6-keto-PGF,, production by cultured endothelial cells compared with age-matched control subjects (2% PDS: 117.8 f 4.3 pg/104 cells/h vs. 150.3 f 10.3; 5% PDS: 183.3 + 5.9 vs. 219.0 f 6.1; 10% PDS: 185.8 + 8.7 vs. 391.4 + 17.0). It was evident that PDS obtained from diabetic patients contained less stimulatory activity on 6-keto-PGF,, production. Fig. 3 indicates that PDS obtained from aged patients with atherosclerotic disease also showed a significant reduction in the stimulation of 6-keto-PGF,, production at final concentrations of 5% and 10% compared with young healthy controls (5% PDS: 184.2 f 11.2 pg/104 cells/h vs 408.2 f 14.3; 10% PDS: 363.6 f 7.2 vs 428.0 f 24.0). It was evident

Fig. 3. Comparison of 6-keto-PGF,, production after addition of PDS from aged patients with atherosclerotic disease and young healthy controls. Results are shown as mean + SE. Significantly different, * P < 0.05, * * P c 0.001.

Pg/lo’ cells/hour

y P
C DM Young

Middle

Old

Fig. 4. Comparison of 6-keto-PGF,, production after addition of 10% pooled PDS from young (n = 10) and middle aged controls (C, n = 10). patients with NIDDM (DM, n = 11) and old, aged patients with atherosclerotic disease (n = 10). Results are shown as mean + SE of 4 experiments. Significantly different, * P -z0.05,** P-c 0.001.

65 that PDS from aged patients with atherosclerotic disease contained less stimulatory activity on 6production. Finally, 6-keto-PGF,, keto-PGF,, production stimulated by 10% PDS was compared among the 4 groups listed in Table 1. Pooled PDS was prepared from each group: patients with NIDDM (DM, n = 11) ranging in age from 32 to 62 years (43 + 4, mean + SE), age-matched controls (C, n = 10) ranging from 31 to 56 years (41 f 3) aged patients with atherosclerotic disease (n = 10) ranging from 72 to 101 years (81 + 3) and young healthy controls (n = 10) ranging from 24 to 27 years (25 + 1). The results are shown in Fig. 4. Namely, 6-keto-PGF,, production stimulated by 10% PDS from patients with NIDDM or aged patients with atherosclerosis was significantly reduced compared with age-matched controls or young healthy controls, respectively. It was evident that the PDS stimulatory activity on 6-ketoPGFim production was reduced in diabetics and aged patients with atherosclerosis. It was interesting that 10% PDS from patients with NIDDM showed significantly less stimulatory activity on 6-keto-PGFiu production than even aged patients with atherosclerosis. Discussion Prostacyclin (PGI,) generated by vascular endothelial cells is a potent prostanoid which inhibits platelet adhesion and aggregation, and therefore prevents the development of vascular lesions such as atherosclerosis. Recent studies demonstrated that PGI, generation by the vascular endothelium was reduced in human arteriosclerotic tissues [l], while the circulating level of 6keto-PGF,, decreased with aging [2] and in patients with cerebrovascular disease [8], ischemic heart disease [9] or diabetes mellitus associated with diabetic vascular complications [lo-121. In experimental animals, PGI, synthesis was also reduced in atherosclerotic arteries from rabbits [ 131 and in the aortic ring obtained from aged [14] or streptozotocin-induced diabetic rats [5]. These reports strongly suggested that reduced PGI, generation by the vascular wall may be closely related with the pathogenesis of vascular lesions such as atherosclerosis in aged humans as well as diabetic patients.

On the other hand, Maclntyre et al. [3] found that human plasma contained a stimulatory activity on PGI, production by cultured endothelial cells. It was suggested that the activity came from a specific stimulant which was heat labile, nondialyzable, and resistant to freezing and thawing. In the present study, plasma-derived serum (PDS) obtained from human subjects stimulated PGI, production by cultured bovine aortic endothelial cells in a time- and dose-dependent manner. In addition, this stimulation of PGI, production by PDS was enhanced by arachidonic acid but inhibited by aspirin. These results suggest that human PDS contains a stimulatory activity on PGI, production by cultured endothelial cells. The PDS stimulatory activity on PGI, production decreased with aging, and PDS obtained from aged patients with atherosclerotic disease showed significantly less activity than that from young healthy controls. Furthermore, PDS obtained from patients with NIDDM also showed significantly less activity than that from age-matched controls. Reduced stimulatory activity in PDS was observed even in early diagnosed NIDDM without vascular complications [4]. Our previous study [5] demonstrated that PDS obtained from streptozotocin-induced diabetic rats also showed significantly less activity on PGI, production by cultured bovine aortic endothelial cells. These findings suggest that the stimulatory activity in serum may be important in the regulation of PGI, synthesis in the vascular wall, and the reduced stimulatory activity may depress PGI, generation by the vascular wall, leading to the development of atherosclerosis in aged humans and patients with NIDDM. However, Aanderud et al. [15] demonstrated that whole blood serum obtained from insulin-dependent diabetic patients inhibited PGI, production by cultured human umbilical endothelium. Our present study suggests that a specific inhibitor of PGI, production may be present in diabetic serum. However, it can not be ruled out that unknown inhibitory factor(s) on PGI, production might appear and/or increase in PDS from NIDDM and aged patients with atherosclerosis. Coughlin et al. [16] demonstrated that a stimulatory activity on PGI, production in serum came from platelets during aggregation, suggesting that this activity was probably due to platelet-derived growth fac-

66 tor (PDGF). To exclude platelet factors, PDS was prepared from platelet poor plasma. Ritter et al. [17] also found a dialyzable factor stimulating PGI, production in human serum. Our preliminary data suggested that this stimulatory activity in the serum may be a protein (app. molecular weight 16000) which is relatively heat stable (56“ C, 30 min), nondialyzable, and resistant to freezing and thawing [4,5]. In any case, further study is needed to purify and characterize this stimulatory activity in PDS. In conclusion, plasma-derived serum contained an activity which stimulated PGI, production by cultured bovine aortic endothelial cells, and this activity was reduced in diabetic and aged patients with atherosclerotic disease. From these results, it was suggested that reduced circulating stimulatory activity on PGI, production by the vascular wall may be one of the pathogenic mechanisms behind the development of vascular lesions such as atherosclerosis. Acknowledgment This work was supported by a Grant-in-Aid for Scientific Research (Grant No. 462180150431) from the Ministry of Education, Science and Culture, Japan. References 1 Sinzinger, H., Feigl, W. and Silberbauer, K., Prostacyclin generation in atherosclerotic arteries, Lancet, ii (1979) 469. 2 Masotti, G., Poggesi, L., Galanti, G., Trottf, F. and Neri Serneri, G.G., Prostacyclin production in man. In: Lewis, P.J. and G’Grady, J. (Eds.), Clinical Pharmacology of Prostacyclin, Raven Press, New York, 1981 p. 9. 3 MacIntyre, D.E., Pearson, J.D. and Gordon, J.L., Localization and stimulation of prostacyclin production in vascular cells, Nature, 271 (1978) 549. 4 Inoguchi, T., Umeda, F., Watanabe, J. and Ibayashi, H., Reduced serum-stimulatory activity on prostacyclin production by cultured aortic endothehal cells in diabetes mellitus, Haemostasis, 16 (1986) 447.

5 Inoguchi, T., Umeda, F., Watanabe, J. and Ibayashi, H., Stimulatory activity on prostacyclin production decreases in sera from streptozotocin-induced diabetic rats, Diabetes Res. Clin. Pratt., 3 (1987) 243. 6 Pledger, W.J., Stiles, C.D., Antoniades, H.N. and Scher, C.D., Induction of DNA synthesis in Balb/c 3T3 cells by serum components: reevaluation of the commitment process, Proc. Natl. Acad. Sci. USA, 74 (1977) 4481. 7 Jaffe, B.M., Behrman, H.R. and Parker, C.W., Radioimmunoassay measurement of prostaglandins E, A, and F in human plasma, J. Clin. Invest. 52 (1973) 398. 8 Uyama, O., Nagatsuka, K., Nakamura, M., Matsumoto, M., Fujisawa, A., Yoneda, S., Kimura, K. and Abe, H., Plasma concentrations of 6-keto-prostaglandin Fi, in patients with hypertension, cerebrovascular disease or Takayasu’s arteritis, Thromb. Res., 25 (1982) 71. 9 Inoue, T., Measured effects and clinical correlations of antiplatelet agents on plasma levels of thromboxane A, and 6-keto-prostaglandin F,, and fatty acid composition of platelets in ischemic heart disease and cerebral infarction, J. Jap. Geriatr. Med., 20 (1983) 294. 10 Dollery, C.T., Friedman, L.A., Hensby, C.N., Kohner, E., Lewis, P.J., Porta, M. and Webster, J., Circulating prostacyclin may be reduced in diabetes. Lancet, ii (1979) 1365. 11 Silberbauer, K., Schemthaner, G., Sinzinger, H., PizaKatzer, H. and Winter, M., Decreased vascular prostacyclin in juvenile-onset diabetes, N. Engl. J. Med., 300 (1979) 366. 12 Watanabe, J., Umeda, F., Sugimoto, H., Wasada, T. and Ibayashi, H., Decreased prostacyclin production and platelet abnormalities in diabetes mellitus, J. Jap. Diabet. Sot., 28 (1985) 1229. 13 Dembinska-Kiec, A., Gryglewski, T., Zmuda, A. and Gryglewski, R.J., The generation of prostacyclin by arteries and by the coronary vascular bed is reduced in experimental atherosclerosis in rabbits, Prostaglandins, 14 (1977) 1025. 14 Chang, W.C. and Tai, H.H., Changes in arachidonate metabolism in aortas and platelets in aging rats, Prostaglandins Leukotrienes Med., 12 (1983) 149. 15 Aanderud, S., Krane, K. and Nordy, A., Influence of glucose, insulin and sera from diabetic patients on the prostacyclin synthesis in vitro in cultured human endothelial cells, Diabetologia, 28 (1985) 641. 16 Coughlin, S.R., Moskowitz, M.A., Zetter, B.R., Antoniades, H.N. and Levine, L., Platelet-dependent stimulation of prostacyclin synthesis by platelet-derived growth factor, Nature, 288 (1980) 600. 17 Ritter, J.M., Ongari, M.A., Orchard, M.A. and Lewis, P.J., Prostacyclin synthesis is stimulated by a serum factor formed during coagulation, Thrombos. Haemostas., 49 (1983) 58.