Reconstitution of the vascular wall in vitro

Experimental Cell Research 162 (1986) 151-158

Reconstitution A Novel Model

of the Vascular

to Study Interactions Smooth Muscle

Wall In Vitro

between Cells

Endothelial

and

M. F. van BUUL-WORTELBOER,’ H. J. M. BRINKMAN,’ K. P. DINGEMANS,* Ph. G. DE GROOT,3 W. G. van AKEN’ and J. A. van MOURIK’, * ‘Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, Amsterdam, ‘Department of Pathology, Academic Medical Centre, Amsterdam, and ‘Department of Haematology, University Hospital, Utrecht, The Netherlands

To study the biology of the endothelium under conditions that mimic the architecture of the vessel waU, endothelhd cells were grown on a collagen lattice containing a multilayer of smooth muscle cells. Light and electron microscopy of such cqhures revealed a confluent monolayer of flattened endothelial cells. In co-culture, endothelial cells tend to elongate, whereas in the absence of smooth muscle cells, the endothelial cells show the polygonal morphology typical for cultures of endothelial cells grown on polystyrene substrates. As conditioned culture media of endothelial cells contain substances that may both promote or inhibit the growth of smooth muscle cells, the availability of this vessel wall model prompted us to examine to what extent endothelial cells regulate the proliferation of smooth muscle cells when these cells are maintained in co-culture. Here we show that endothelial cells suppress the proliferation of co-existing smooth muscle cells. This finding suggests that under physiological conditions the balance of the action of growth-promoting and growth-inhibiting substances produced by endothelial cells is in favour of the latter. @19%6 Academic Rcss. Inc.

Cultures of endothelial cells seeded on solid impermeable substrates have proven useful for the study of several processes implicated in the physiology of the vessel wall including biosynthesis of prostaglandins [ 11, matrix and plasma proteins [2-71 or binding of blood components to the endothelial surface [8-151. However, unlike the situation in vivo, such cultures lack the abluminal space and consequently no longer form a partition that may mediate transport of tissue and blood components. Therefore, endothelial cell-mediated processesthat are representative for the intact vessel wall cannot be fully studied with such isolated cultures of endothelial cells. So far, attempts to study these processes in detail have suffered from the inability to construct a culture system that mimics the anatomy of the vessel wall. Here we describe a method for the culture of endothelial cells on a loose collagen matrix that contains smooth muscle cells: a co-culture that approximates * To whom otfprint requests should be sent. Address: c/o Publication Secretariat, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, P. 0. Box 9406, 1006AK Amsterdam, The Netherlands. Copyright @ 1986 by Academic FTCSS, Inc. AU tights of reproduction in any form reserved CI3144327/U6 $03.00

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the architecture of the vessel wall. We show that such a reconstituted vessel wall allows study of endothelial cell-mediated processes that govern the growth of smooth muscle cells. This question seems of particular importance because smooth muscle cell proliferation is thought to be one of the main events implicated in the pathogenesis of atherosclerotic lesions [16, 171. Our data indicate that the proliferative response of quiescent smooth muscle cells to whole blood serum is inhibited by endothelial cells when these cells are assembled in co-culture. We propose that in vivo endothelial cells may hinder the ability of smooth muscle cells to proliferate upon exposure to exogenous stimuli. MATERIALS

AND METHODS

Cell Culture Co-cultures of endothelial cells and smooth muscle cells were prepared as follows. Calf-skin collagen (Sigma Chemical Co., St. Louis, MO., type- III) was dissolved to 0.2% (w/v) in 0.5 M acetic acid for 24 h at 4°C under continuous stirring and thereafter dialysed against phosphate-buffered saline (PBS, pH 7.4) and subsequently against RPMI/M199 (1 : 1 (v/v); Gibco, Paisley, UK). The collagen solution was sterilized for 30 min with ultraviolet light at 4”C, and 0.3 ml was allowed to form a gel for 15 min at 37°C in plastic culture wells of 16 mm diameter. Cultured smooth muscle cells, derived from explants of human umbilical arteries [18], were seeded on the collagen gel in RPMI/M199 supplemented with 20% (v/v) human whole blood serum (WBS). After 24 h, the culture medium was removed, and the smooth muscle cells were covered with 0.2 ml of the 0.2% collagen solution. AAer gelation of collagen, cultured endothelial cells, isolated from human umbilical veins and subcultured [19], were seeded (14x lo4 cells/cm2) on top of the gel in RPMI/M199 supplemented with 20% WBS.

Microscopy Silver nitrate staining of endothelial cells was performed according to a modification of the method of Poole et al. [ZO] as described by Furie et al. [21]. For transmission electron microscopy (TEM), cultures were fixed in 2 % glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4, post-fixed in 1% 0~01 in 0.1 M cacodylate, pH 7.4, stained with 1% uranyl acetate, dehydrated and embedded. The sections were stained with many1 acetate and lead citrate before examination by electron microscopy. For scanning electron microscopy (SEM), cultures fixed in 2 % glutaraidehyde and post-fixed in 1% 0~0, were dehydrated and critical point-dried from liquid C02. The dried collagen gels were attached to ahnninium slugs with double-sided adhesive tape and the samples were coated with gold in a sputtering device.

Measurement of Proliferation Proliferation of cells was assessed by either cell counting or by measuring [3H]thymidine incorpora-

tion. In caseof cell counting,endothelialcells and smooth muscle cells were harvested separately by incubation with 10 U of collagenase per ml (Worthington B&hem. Corp., Freehold, N.J., CLSA) in PBS (PH 7.4) during 5 min (endothelial cells) and 15-25 min (smooth muscle cells), respectively, and subsequent centrifugation for 2 min at 8O@lg. The pellets were resuspended in Vypsin-EDTA (G&o, Paisley, Scotland) and the cells were counted in a hemocytometer. [‘HlThymidine incorporation by synchronized smooth muscle cells was measured as follows. Smooth muscle cells were seeded on the bottom of a 5 mm diameter well (about 104 cells/well). Synchronized,quiescentsmoothmusclecells were obtained by maintaining the cells for 3 days in RpMyM199 supplemented with 0.4% WBS. Smooth muscle cells were then covered with soluble collagen (50 ~0. After gelation, 0.4,20 or 20% WBS together with human endothelial cells (0.75, 1.5,3 or 6~ 104cells/cm2) were layered on top of the gel and [3H]thymidine (0.2 ~Ci/well; The RadiochemiCd Centre, Amersham, Bucks, England) incorporation was measured for 24 h in both endothefid and smooth muscle cells. Endothelial and smooth muscle cells were harvested separ&ly after incubation with collagenase with a Titertek Harvester, as previously described [22]. EXP Cell Res 162 (I%?b)

Reconstitution of the vascular

wall in vitro

153

Fig. 1. SEM of endothelial cells, co-cultivated with smooth muscle cells for 4 days. x40.

RESULTS Morphology Endothelial cells form a confluent monolayer both when seeded on collagen gel only and on smooth muscle cells covered with collagen gel (fig. 1). In co-culture with smooth muscle cells, the endothelial cells elongate, whereas endothelial cells grown in the absence of smooth muscle cells show the characteristic polygonal morphology (fg 2a, b). Smooth muscle cells tend to migrate into the gel in various directions (fig. 2 c). This behaviour is not affected by the presence of endothelial cells. Endothelial cells do not migrate into the gel. TEM and light microscopy of transverse sections of collagen gels with both endothelial and smooth muscle cells also show a continuous monolayer of flattened endothelial cells and a multilayer of smooth muscle cells (fig. 3). The cocultures remain stable for more than 10 days. Thereafter, dissolution of the collagen gel may occur, probably due to the collagenolytic activity of smooth muscle cells [23]. Collagen lysis is not observed when endothelial cells are cultured alone. Cell Growth The proliferation rate of endothelial cells in co-culture is about the same as that of endothelial cells cultured alone on collagen gels (fig. 4a) or on fibronectincoated dishes (not shown). The growth rate of smooth muscle cells in the presence of endothelial cells, however, is lower than that of smooth muscle cells cultured in collagen gels in the absence of endothelial cells (fig. 4a) or smooth muscle cells cultured on fibronectin-coated dishes (not shown). These data represent the growth of smooth muscle cells that were maintained under growthstimulating conditions. We have also studied the influence of endothelial cells on

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Fig. 2. (a, b) Phase contrast micrographs of endothelial cells stained with silver nitrate. The endothelial cells were cultured on top of a collagen gel in (II) absence; or (b) presence of smooth

muscle cells. (c) Phase-contrast micrograph of smooth muscle cells migrating from a smooth muscle cell layer (out of focus) into the collagen gel. Fig. 3. (a) Light micrograph of a transverse section of endothelial cells (EC) and smooth muscle cells (SMC) co-cultivated for 4 days. (6, c) TEM of endothelial cells co-cultivated with smooth muscle Exp Cell Res 162 (19%)

Reconstitution of the vascular wall in vitro

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cloys in culture

Fig. 4. (a) Proliferation of 0, endothelial cells, and 0, smooth muscle cells in -, co-culture; or --cultured separately in BPMIh4199 supplemented with 20% WBS. Each point represents the meak of three determinations k SD. (b) Inhibition of [3Hlthymidine uptake by synchronized smooth muscle ceils by endothelial cells upon stimulation with 20% WBS (see Materials and Methods). Each histogram represents the mean of six determinations f SD.

the initiation of proliferation of growth-arrested smooth muscle cells, a condition more representative for the situation in vivo [24]. Upon stimulation by serum, synchronized smooth muscle cells in co-culture with endothelial cells incorporate less L3H]thymidine than do smooth muscle cells in the absence of endothelial cells (fig. 4 b). This effect is dependent on the number of endothelial cells seeded on top of the collagen matrix (fig. 4 b). Both confluent (6x lo4 cells/cm’) and subconfluent endothelial ceil monolayers were able to inhibit [3Hlthymidine incorporation in smooth muscle cells. However, there seems to be no clear relationship between the amount of inhibitory activity of endothelial cells on smooth muscle cell proliferation and the growth state of endothelial cells. Similarly, the increase in the number of smooth muscle cells upon stimulation was inhibited by endothelial cells. To rule out the possibility that the observed inhibition of growth of smooth muscle cells is caused by an impaired availability of growth factors and nutrients cells. The lateral borders of adjacent endothelial cells are always in close contact, forming cell junctions often with the characteristics of tight junctions (b). The cytoplasm of endothelial cells (c) contains many organelles, especially rough endoplasmic reticulum (RER), pinocytotic vesicles (6, arrowhead), mitochondria, Golgi apparatus and occasional Weibel-Palade bodies. The cytoplasm of endotbelial cells also contain microtubuli, intermediate filaments and microtilaments (MF). (4 Smooth muscle ceils. These cells, too, contain many organelles. MicroSlaments (MF) are generally present as discrete bundles along the cell periphery. (a) x500; (b) X29000; (c) X22500; (4 X6500. Exp Cell Res 162 (1986)

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scavenged by endothelial cells, the co-culture experiments were repeated with synchronized smooth muscle cells cultured on polycarbonate filters (Nucleopore Corp., Pleasanton, Calif.; 8 uM pore), attached to a plastic ring and placed in the culture medium, thereby providing smooth muscle cells directly (from beneath through the filter) with nutrients and r3H]thymidine. The results were similar to the previous data: in the presence of endothelial cells, synchronized smooth muscle cells incorporate about 6&70% less [3H]thymidine in response to serum than do smooth muscle cells covered with collagen gel without endothelial cells on top of it. The [3H]thymidine uptake by endothelial cells is not affected by smooth muscle cells (not shown). It seems likely, therefore, that, at least in coculture, endothelial cells suppress the growth of smooth muscle cells. DISCUSSION The present study raises an important issue: to what extent do endothelial cells regulate the growth of smooth muscle cells in situ? In the normal non-injured vessel, smooth muscle cells have a very low mitotic rate 1251. However, in response to repeated vascular injuries, these cells may undergo a phenotypic change, start to migrate and proliferate at the site of the vascular lesions [26,27]. This process is considered to be a reaction to the disturbance of a delicate balance in the micro-environment of the vessel wall, initiated by adhesion and aggregation of blood platelets and subsequent release of platelet-derived growth factor (PDGF) at sites of vascular injury [16, 171. It is also thought that vesselwall cells themselves may regulate smooth muscle cell growth. Evidence has been provided that endothelial cells are able to produce factors that either inhibit [28-301 or stimulate [31-341 smooth muscle cell growth. Most of these data were derived from experiments designed to study the effect of conditioned media of endothelial cells, or crude fractions thereof, on the proliferation of connective tissue cells. Short-living (secretion) products of endothelial cells, which also may exert a regulatory effect on smooth muscle cell proliferation, could be overlooked in this way. In addition, it may be possible that smooth muscle cells provide precursors or induce or potentiate the production of endothelial cell-derived regulator(s) of smooth muscle cell growth. Our study provides evidence that the inhibitory activity of both confluent and subconfluent endothelial cells upon the initiation of proliferation of smooth muscle cells is predominant when smooth muscle cells are kept in co-culture under physiological conditions, conditions in which (unstable) endothelial cell secretion products, either from the luminal or abluminal site, can interact directly with smooth muscle cells. We were never able to detect any endothelial cellderived growth-stimulatory effect on smooth muscle cell proliferation. Davies et al. [31] examined a co-culture system with bovine aortic endothelial cells cultured on microcarriers separated from a smooth muscle cell layer by a filter. Under fip Cell Res 162 (1986)

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these conditions, endothelial cells seem to enhance smooth muscle cell proliferation. How to explain these opposite effects is not clear at the moment. These differences could, at least in part, stem from differences in species studied (human vs bovine) or differences in growth media [28]. Even more important could be the difference in the design of the co-culture system. One unexpected finding is that endothelial cells tend to elongate only when they grow in the presence of smooth muscle cells. This behaviour suggeststhat not only blood flow is responsible for the elongated shape of endothelial cells in vivo [35]. Perhaps this change of cellular shape is due to an altered composition of the collagen matrix [36] as a result of the secretion of matrix proteins [24] or collagenolytic activity produced by smooth muscle cells [23]. The co-culture technique described in this study offers many possibilities for a more complete analysis of endothelial cell function. The endothelial cell layer can be removed selectively from the collagen matrix without disturbing the smooth muscle cell layer, and samplesfrom both the luminal and subendothelial compartment can be taken by collecting the medium and subsequent centrifugation of the collagen gel, thus allowing studies on endothelial cell-mediated transport phenomena either in the presence or absence of smooth muscle cells. In addition, with the present design, many variables (e.g., dimensions, vasoactive agents, stimuli, different collagen lattices) can be introduced in the experimental protocols. Therefore, this reconstituted vessel wall may be useful not only to study endothelial-smooth muscle cell interactions but also other properties known to be associated with the endothelium, as the blood-tissue barrier. The authors wish to thank Dr N. Out and T. M. Rolf for their assistance with the SEM and TEM. This study was supported by the Netherlands Heart Foundation (grant no. 28.004).

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