Atherosclerosis, 94 ( 1992) 89-9 1 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/%05.00

89

Printed and Published in Ireland

ATHERO 04829

Letter to the Editors

Endothelial cell damage and homocysteine A.D. Blann Dept. of Surgery, University Hospital of South Manchester. Nell Lane. West Didsbury. Manchester M20 8LR (UK)

(Received 11 February, 1992) (Revised, received 2 March, 1992) (Accepted 17 March, 1992)

Key words: Von Willebrand factor antigen; Atherosclerosis;

Dear Editors,

The proposed relationship between homocysteine and atherosclerosis is well established through both animal and clinical studies although the precise mechanism has not been fully elucidated [1,2]. One popular hypothesis is that of endothelial cell (EC) injury [3,4], a theory recently supported by the work of Dudman et al. [5], who, extending previous in vitro data [3], showed that sulphydryl-containing amino acids (homocysteine and cysteine) induce the detachment of EC from their culture dishes. Increased levels of von Willebrand factor antigen (vWFAg, a specific product of the endothelium) are found where there is injury/damage to the vasculature as occurs in inflammatory vascular disease and in atherosclerosis and its major risk factors [6,7]. In order to test the effect of homocysteine on the vasculature, the culture medium of radiolabelled human umbilical vein EC in vitro Correspondence to: A.D. Blann, Dept. of Surgery, University Hospital of South Manchester, Nell Lane, West D&bury, Manchester M20 8LR, UK. Tel.: 061-445 8111 Ext. 3840; Fax: 061-447 3846.

Homocysteine; Cytotoxicity

was supplemented with varying concentrations of the amino acid in a manner analogous to that of Wall et al. [3]. Briefly, EC were obtained from the lumen of umbilical veins by digestion with 0.1% collagenase (Sigma) at 37°C for 15 min [8,9]. Cells were flushed out with saline, washed and grown in standard tissue culture techniques in RPM1 plus 20% fetal calf serum, antibiotics and glutamine. When confluent, cells were removed from culture with trypsin/EDTA and washed down to a 1 ml volume in serum-free RPMI. They were radiolabelled with 3.7 MBq “‘indium (Amersham) at room temperature for 4 min, washed free of unbound label and dispensed to a microtitre plate at lo6 cells/ml in RPMI. RPM1 (100 ~1) supplemented with varying concentrations of homocysteine (Sigma) was added to 100 p cell suspension. After 3 h, the supernatant was recovered and tested for the proportion of released isotope relative to positive (= release from cells cultured with Triton X-100) and negative (= release from cells cultured with RPM1 alone) controls (= % cytotoxicity). The supematant was then tested by a standard ELISA method for vWFAg (Fig. 1).

90

15 EC

vWFAg (NJ d/L) -

% 10

20

d.1

cl.3

20

Homocystaina

Fig. I. Cytotoxic

and vWFAg-inducing

action

of homocysteine.

There was a clear dose-related cytotoxicity of homocysteine towards EC which was evident at concentrations as low as 0.1 mmol/l (closed circles). However, the ability of homocysteine to promote release of vWFAg was apparent only when the concentration of the homocysteine was 1 mmol/l and 3 mmol/l (open circles, P < 0.05), with a greater increase at 10 mmol/l and 20 mmol/l (P < 0.01). This data, which supports the work of Wall et al., would seem to suggest that high levels of homocysteine are cytotoxic to EC and also promote the release of vWFAg. The latter molecule is not merely a marker of EC injury but may have a more active role in atherosclerosis as it can agglutinate platelets and promote their adhesion to the subendothelium [lo]. Since homocysteinaemia is often present in ischaemic or premature coronary artery disease and in peripheral occlusive artery disease [ 1 l- 131, there may also be high levels of circulating vWFAg. This can be tested by measuring paired levels of vWFAg and homocysteine in patients with atherosclerosis. A strong correlation between

(mmol/L)

Secretion

of vWFAg

(open circles) and cytotoxicity

(closed circles).

these indices (in the absence of other risk factors which also increase vWFAg [6,7]) would be evidence in favour of this hypothesis. Someone should do this experiment. References Harker, L.A., Harlan, J.M. and Ross, R., Effect of suhinpyrazone on homocysteine-induced endothelial injury and arteriosclerosis in baboons, Circ. Res., 53 (1983) 731. McCully, KS. and Vezeridis, M.P., Homocysteine thiolactone in arteriosclerosis and cancer, Res. Commun. Chem. Path. Pharm., 59 (1988) 107. Wall, R.T., Harlan, J.M., Harker, L.A. and Striker, G.E., Homocysteine-induced endothelial cell injury in vitro: A model for the study of vascular injury, Thromb. Res., 18 (1980) 113. Ross, R., The pathogenesis of atherosclerosis: N. Engl. J. Med., 314 (1986) 488.

An update,

Dudman, N.P.B., Hicks, C., Wang, J. and Wilcken, D.E.L., Human arterial endothelial cell detachment in vitro: its promotion by homocysteine and cysteine, Atherosclerosis, 91 (1991) 77. Blann, A.D., Hopkins, J., Winkles, J. and Wainwright, A.C., Serum and plasma von Willebrand factor antigen concentrations in connective tissue disorders, Ann. Clin. B&hem., 29 (1992) 67.

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8

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Blann, A.D. and McCollum, C.N., Haemostatic risk factors in patients with vascular disease. Atherosclerosis, 93 (1992) 255. Jaffe. EA., Nachman, R.L., Becker, CC., Minick, C.R., Culture of human endothehal cells derived from umbilical veins. J. Clin. Invest., 52 (1973) 2745. Blann. A.D., Garner, C.M. lndium 11I (“‘In) as a label for endothelial cells and fibroblasts used as target cells in cytotoxicity tests. Med. Lab. Sci., 42 (1985) 92. Wagner, D.D., Cell biology of von Willebrand factor, Annu. Rev. Cell Biol., 6 (1990) 217.

1I

Genest.

12

Prevalence of familial hyperhomocyst(e)inemia in men with premature coronary artery disease. Arterioscler. Thromb., 11 (1991) 1129. Malinow, M.R., Kang, S.S., Taylor, L.M. et al..

13

J.J.,

McNamara,

J.R.,

Upson.

B.

et

al.,

Prevalence of hyperhomocyst(e)inaemia in patients with peripheral arterial occlusive disease, Circulation, 79 (1989) 1180. Olszewski, A.J. and Szostak, W.B., Homocysteine content of plasma proteins in ischaemic heart disease, Atherosclerosis, 69 (1988) 109.

Endothelial cell damage and homocysteine.

Atherosclerosis, 94 ( 1992) 89-9 1 0 1992 Elsevier Scientific Publishers Ireland, Ltd. All rights reserved. 0021-9150/92/%05.00 89 Printed and Publi...
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