Atherosclerosis, 83 (1990) 9-14 Elsevier Scientific Publishers Ireland, Ltd.

ATHERO

04485

Effects of fibrinogen and fibrin on the migration of vascular smooth muscle cells in vitro Michitaka Naito, Toshio Hayashi, Masafumi Kuzuya, Chiaki Funaki, Kanichi Asai and Fumio Kuzuya Department

of Geriatrics, Nagoya Uniuersity School of Medicine, Nagoya (Japan) (Received 11 December, 1989) (Revised, received 21 February, 1990) (Accepted 22 February, 1990)

Summary The migration of vascular smooth muscle cells from the media into the intima and their proliferation in the intima play an important role in the pathogenesis of atherosclerosis. We examined the effects of fibrinogen and fibrin on the migration of cultured bovine aortic smooth muscle cells using a modified Boyden chamber assay. The cells migrated to a gradient of soluble fibrinogen. Checkerboard analysis indicated that the effect was largely directional in nature (chemotaxis). The cells also migrated in a dose-dependent manner to a gradient of substrate-bound fibrinogen (haptotaxis). Fibrin, converted from substrate-bound fibrinogen by thrombin, also induced haptotaxis of smooth muscle cells. These observations suggest that, by recruiting smooth muscle cells from the media into the intima, fibrinogen and fibrin may be involved in the pathogenesis of arterial intimal thickening, atherosclerosis, and the organization of a thrombus.

Key words:

Atherosclerosis; Fibrinogen; Haptotaxis; Pathogenesis

Fibrin;

Introduction It has been proposed that the directional migration of vascular smooth muscle cells (SMCs) from the media into the intima and their proliferation in the intima may be the main process in the pathogenesis of atherosclerosis [l-3]. A key factor

Correspondence to: Dr. M. Naito, Department of Geriatrics, Nagoya University School of Medicine, Nagoya 466, Japan. 0021-9150/90/$03.50

Smooth

muscle

cells;

Cell

migration;

Chemotaxis;

in this process may be platelet-derived growth factor (PDGF), released from platelets, macrophages, endothelial cells, and even from the SMCs themselves [3]. However, other investigators suggest that the clotting system, including fibrinogen and fibrin, is important in the initiation and development of atherosclerosis [4-61. Many morphological or immunochemical studies have demonstrated the presence of fibrinogen and fibrin in atherosclerotic lesions [4,5]. Recently, Bini et al. [7], using monoclonal antibodies, has reported that

0 1990 Elsevier Scientific Publishers Ireland, Ltd

10 the transition of fibrinogen to fibrin within the arterial wall was accompanied by the progression of atherosclerosis. However, there are few studies about the effects of fibrinogen and fibrin on the behavior and function of SMCs [8]. The purpose of this study is to examine the effects of fibrinogen and fibrin on the migration of vascular SMCs using a modified Boyden chamber system. A preliminary report of this experiment appeared elsewhere [9].

Materials and methods Materials Purified bovine fibrinogen was obtained from Calbiochem (U.S.A.). Sodium dodecyl sulfatepolyacrylamide gel electrophoresis demonstrated only a single band. Other materials were purchased from the following sources: bovine serum albumin (BSA, essentially fatty acid- and globulin-free, A0281), bovine plasmin, bovine thrombin (T-7513), and synthetic human fibrinopeptides A and B, all from Sigma (U.S.A.); trypsin from Gibco (U.S.A.); Dulbecco’s modified Eagle’s medium (DMEM) from Nissui (Japan); fetal bovine serum (FBS) from Cell Culture Laboratories (U.S.A.); sodium iodide-125, carrier free, from NEN (U.S.A.). Cell culture Fetal bovine aortic SMCs were cultivated as reported previously 191. Cultures were used within 10 passages for migration assays. Cell migration assays Chemotaxis and haptotaxis were assayed as described previously [9,10]. Single cell suspensions of SMCs were obtained by treatment of cultures with 0.05% trypsin/0.02% EDTA. Harvested cells were washed in serum-free DMEM supplemented with 0.5% BSA (w/v) and were resuspended in this medium. Migration assays were performed using 48-well micro-chemotaxis chambers (Neuro Probe, U.S.A.). The lower wells, containing 27 ~1 of the solution to be assayed, were covered with a S-pm pore size polyvinylpyrrolidone (PVP)-free polycarbonate filter (Nucleopore, U.S.A.), and the resuspended cells were added to the upper wells at

a volume of 45 ,ul (2.5 x lo4 cells/well). After 6-h incubation in a CO, incubator (37 o C, 95% sir/5% CO,, humidity 100%) the filter was removed from the chamber and the non-migrated cells were scraped from the upper surface with a wiper blade (Neuro Probe, U.S.A.). Migrated cells on the lower surface were then fixed, stained with Giemsa, and counted under a high power (400 x ) field (HPF). 4 HPFs were counted per sample and averaged.

Coating of filters with fibrinogen or fibrin For the evaluation of haptotaxis. filters were coated by the method of McCarthy et al. Ill] as follows. PVP-free polycarbonate filters were coated on the lower or upper surfaces only by floating filters or on both surfaces by immersing filters in a solution of fibrinogen in BSA-free or 0.5% BSAcontaining DMEM, pH 7.4, overnight at 37” C. Control filters were treated with BSA-free or 0.5% BSA-containing DMEM in the absence of fibrinogen. Fibrin coating was performed by converting coated fibrinogen to fibrin, by treating the fibrinogen-coated filters with 0.25 NIHU/ml thrombin-containing DMEM, pH 7.4, at 37 o C for 6 h. The precoated filters were washed extensively in Dulbecco’s phosphate-buffered saline (PBS) prior to use. Chambers were then assembled with 0.5% BSA-containing DMEM.

Labeling of fibrinogen with ’“‘1 Fibrinogen was radiolabeled with ‘25l using a modified chloramine-T method [12,13]. The “‘Iprotein was greater than 95% precipitable in 20% trichloroacetic acid.

Binding of fibrinogen to filters Filters, l-cm’, were floated on the lower surfaces in 0.3 ml 10 pg/ml ‘251-fibrinogen in BSA-free or 0.5% BSA-containing DMEM, pH 7.4, in a 24-well plastic plate (Corning, U.S.A.) at 37 o C overnight. After excessive washing in PBS, the radioactivity associated with the filters was counted.

Statistical analysis Statistical analysis t-test. A P value of significant.

was performed by Student’s < 0.05 was considered to be

11

Results

‘251-fibrinogen bound(%)

Migration of SMCs to soluble and substrate-bound fibrinogen Fibrinogen stimulated the migration of SMCs in the modified Boyden chamber assay (Fig. 1A). When a series of fibrinogen concentrations was introduced into the lower wells, migration was stimulated in a dose-dependent manner. Checkerboard analysis indicated that the migration was mainly directional (chemotaxis) [9]. SMCs also migrated over substrate-bound fibrinogen in the absence of soluble fibrinogen 100

DMEM+O.S%BSA II

DMEM+S%BSA &

to filters. The binding of I$g. 2. Binding of ‘251-fibrinogen I-fibnnogen to filters was measured in BSA-free or 0.5% or 5% BSA-containing DMEM as described in Materials and Methods. Results are expressed as mean + SD, n = 4, * P c

A _L

T I

0’ F~brmogen(~g/ml) I 0

I

1

I

100

1000

loo-

_

“u 50m al ;;i .!2 I DMEM+0,5%BSA x

O-

* I-*

O+;

0.001.

1

f

k I si =

DMEM

P

Fig. 1. Dose-response curves for chemotactic (A) and haptotactic (B) responses of SMCs to fibrinogen. (A) Increasing concentrations of fibrinogen were added to the lower wells, diluted in DMEM supplemented with 0.5% BSA in a volume of 27 al. The chambers were then assembled with filters, 45 al 5 X 10’ cells/ml in DMEM supplemented with 0.5% BSA was added to the upper wells. Incubation time was 6 h. (B) Filters were precoated with a step gradient of increasing concentrations of fibrinogen in BSA-free (0) or 0.5% BSA-containing (0) DMEM as described in Materials and Methods. 27 pl DMEM supplemented with 0.5% BSA was added to the lower wells; the chambers were assembled with the filters, and 45 al 5 X lo5 cells/ml in DMEM supplemented with 0.5% BSA was added to the upper wells. Incubation time was 6 h. Data are expressed as mean _+SD, n = 12.

(Fig. 1B). The response increased in a dose-dependent manner with the peak response zct 10 pg/ml. When the fibrinogen-precoating procedure was performed with BSA-free DMEM, fibrinogen was effectively coated on the filter. However, when the procedure was performed with 0.5% BSA-containing DMEM, the binding was very low and the migration of SMCs was not stimulated (Figs. 1B and 2). These results indicate that the stimulatory effect of soluble fibrinogen in Fig. 1A. performed with 0.5% BSA-containing DMEM, is mainly chemotactic and not haptotactic. Analysis of the response of SMCs to substrate-bound fibrinogen Significant migration was observed when the filter was coated on its lower surface with fibrinogen (10 pg/ml). The level of migration was reduced by 60% when the same concentration of fibrinogen was used to coat both the upper and lower filter surfaces, suggesting that the effect is mainly directional (Fig. 3). Specificity of fibrinogen-induced haptotaxis To investigate the specificity of the haptotaxis induced by fibrinogen, we performed the following experiments. Overnight digestion of fibrinogen with trypsin or plasmin completely abolished the haptotactic effect, suggesting that the induction of migration by substrate-bound fibrinogen is in fact attributable to the fibrinogen itself and not to possible contaminants (Fig 4).

12 Fibrinogen upper

coating

Migrated

cells/HPF

Lower

+

-

-

+

-I-

*

1

IO

100

1000

Fibrlnogen/F,brln(~g/ml)

Fig. 5. Haptotaxis induced by fibrinogen (0) and fibrin (0). The lower surface of the filters was coated with fibrinogen or fibrin as described in Materials and Methods. The procedure for the migration assay is described in Fig. lB, n = 8.

+

Fig. 3. Analysis of the response of SMCs to substrate-bound fibrinogen. Filters were precoated with 10 pg/ml fibrinogen in BSA-free DMEM, as described in Materials and Methods, on the upper surface, the lower surface, both surfaces, or neither (control). The procedure for migration assay was performed as described in Fig. 1B. Results are expressed as meanf SD, n=12, *P

Effects of fibrinogen and fibrin on the migration of vascular smooth muscle cells in vitro.

The migration of vascular smooth muscle cells from the media into the intima and their proliferation in the intima play an important role in the patho...
588KB Sizes 0 Downloads 0 Views