EXPERIMENTAL CELL RESEARCH 2 0 0 , 156-167 (1992)

Heparan Sulfate-Degrading Enzymes Induce Modulation of Smooth Muscle Phenotype JULIE H. CAMPBELL,1 ROBYN E. RENNICK, SILVIA G. KALEVITCH, AND GORDON R. CAMPBELL Department o/Anatomy, University of Queensland, St. Lucia, Queensland, 4072, Australia

M a c r o p h a g e s c o c u l t u r e d w i t h r a b b i t a o r t i c smooth muscle cells at a r a t i o of 1:3 d e g r a d e d all the aSS-labeled h e p a r a n sulfate p r o t e o g l y e a n f r o m the s m o o t h muscle s u r f a c e into f r e e sulfate (Kay of 0.84 on 8 e p h a r o s e 6B). Concomitantly, the same m a c r o p h a g e s induced a dec r e a s e in the v o l u m e f r a c t i o n of m y o f i l a m e n t s (Vvmyo) of the smooth muscle cells and a d e c r e a s e in a-actin mRNA as a p e r c e n t a g e of total actin mRNA. Both macrophage lysosomal lysate at n e u t r a l pH and h e p a r i n a s e d e g r a d e d cell-free aSS-labeled m a t r i x deposited b y smooth muscle cells into f r a g m e n t s w h i c h eluted at a Kay of 0 . 6 3 and w h i c h w e r e identified as h e p a r a n sulfate chains by t h e i r complete d e g r a d a t i o n in the presence of low pH n i t r o u s acid. At acid pH the m a c r o p h a g e lysosomal lysate c o m p l e t e l y d e g r a d e d the h e p a r a n sulfate to free sulfate (K~v 0.84). B o t h m a c r o p h a g e lysosomal lysate and c o m m e r c i a l h e p a r i n a s e at n e u t r a l pH induced smooth muscle p h e n o t y p i c change while o t h e r e n z y m e s such as t r y p s i n and c h o n d r o i t i n ABC lyase had n o effect. It was t h e r e f o r e suggested t h a t the active fact o r p r e s e n t in the m a c r o p h a g e s is a lysosomal h e p a r a n s u l f a t e - d e g r a d i n g endoglycosidase (heparinase). Only a small a m o u n t of h e p a r a n s u l f a t e - d e g r a d i n g activity was released into the i n c u b a t i o n m e d i u m b y living macrophages, a n d t h e r e was no h e p a r i n a s e activity on t h e i r isolated plasma m e m b r a n e s , although p r o t e o l y t i c e n z y m e s w e r e evident in both instances. In p u l s e - c h a s e studies, high Vvmyo smooth muscle cells w e r e seen to c o n s t a n t l y i n t e r n a l i z e and d e g r a d e aSS-labeled h e p a r a n sulfate p r o t e o g l y c a n f r o m t h e i r own p e r i c e l l u l a r comp a r t m e n t , suggesting t h a t this m a y be the m e c h a n i s m b y which smooth muscle p h e n o t y p e is m a i n t a i n e d u n d e r n o r m a l c i r c u m s t a n c e s and t h a t r e m o v a l of h e p a r a n sulf a t e f r o m the s u r f a c e of smooth muscle cells and its degr a d a t i o n b y m a c r o p h a g e s t e m p o r a r i l y i n t e r r u p t s this p r o c e s s , i n d u c i n g smooth muscle p h e n o t y p i c change. 9 1992 Academic Press, Inc.

INTRODUCTION T h e r e is now a large body of evidence indicating t h a t s m o o t h muscle cells in t h e i n t i m a of h u m a n arteries 1To whom reprint requests should be addressed. 0014-4827/92 $3.00 Copyright 9 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.

involved in atherogenesis are p h e n o t y p i c a l l y different f r o m t h o s e of t h e u n d e r l y i n g media. T h e s e differences include changes in v o l u m e fraction of m y o f i l a m e n t s (Vvmyo) [1, 2], cell shape [3, 4], actin isoforms [5], myosin isoform [6], t r o p o m y o s i n c o n t e n t [7], i n t e r m e d i a t e filaments [7, 8], c a l d e s m o n [9], m e t a v i n c u l i n [9], cyclic nucleotides [10], P D G F expression [11], fibronectin [12], a n d e x p r e s s i o n of major histocompatibility complexes [13]. Some of these changes m a y play a crucial role in d e v e l o p m e n t or initiation of atherosclerosis [14]. In p r i m a r y cell culture, medial s m o o t h muscle cells which have been e n z y m e - d i s p e r s e d and seeded at densities below confluence undergo similar changes in p h e n o typic expression during the first 5 days a f t e r isolation [15], a c c o m p a n i e d by distinct alterations in biology. In this study we have used the t e r m i n o l o g y of high VvmyO versus low Vvmyo s m o o t h muscle to indicate p h e n o t y p e alterations, r a t h e r t h a n the descriptive t e r m i n o l o g y contractile or synthetic which implies absolute functional changes. P r i o r to change in p h e n o t y p e while the cells have a high Vvmyo as in the n o r m a l vessel wall, the cells can be m a d e to c o n t r a c t with various agonists, do not proliferate logarithmically in response to mitogens f r o m serum, synthesize minimal collagen, and accumulate little lipid even after exposure to high c o n c e n t r a tions of fl-very low density lipoprotein (fl-VLDL) for several days. In contrast, s m o o t h muscle ceils of low Vvmyo do not c o n t r a c t in response to agonists, b u t proliferate logarithmically in response to mitogens f r o m a variety of sources, synthesise 26- to 45-fold the a m o u n t of collagen (particularly collagen type I) a n d accumulate 7-fold the a m o u n t of lipid on exposure to fl-VLDL [16-19]. Since a low Vvmyo, proliferation, synthesis of extracellular m a t r i x (particularly collagen type I), and a c c u m u l a t i o n of lipid are all characteristic features of s m o o t h muscle cells in a t h e r o m a , change in p h e n o t y p e of s m o o t h muscle cells m a y be an i m p o r t a n t initial e v e n t in the d e v e l o p m e n t of this disease. T h i s raises the question, w h a t d e t e r m i n e s the p h e n o t y p i c expression of s m o o t h muscle ceils? In particular, what d e t e r m i n e s w h e t h e r a s m o o t h muscle cell is contractile (high Vvmyo) a n d u n r e s p o n s i v e to atherogenic stimuli such as mitogens a n d certain lipoproteins or w h e t h e r it has a low Vvmyo and is responsive to these agents?

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MODULATION OF SMOOTH MUSCLE PHENOTYPE Studies in our l a b o r a t o r y implicate c e l l - m a t r i x interactions in control of s m o o t h muscle p h e n o t y p e . In prim a r y cell culture, a crude e x t r a c t of g l y c o s a m i n o g l y c a n s f r o m the aortic i n t i m a plus i n n e r media m a i n t a i n s sparsely seeded s m o o t h muscle with a high VvmyO [20]. T r e a t m e n t of this aortic e x t r a c t with h e p a r i n a s e from F l a v o b a c t e r i u m h e p a r i n u m destroys the active factor, indicating t h a t g l y c o s a m i n o g l y c a n s of t h e h e p a r a n sulfate species are responsible; a n d indeed, addition of the closely related g l y c o s a m i n o g l y c a n heparin m a i n t a i n s sparsely seeded s m o o t h muscle with a high Vvmyo [20]. This has been confirmed by H e r b e r t e t al. [21] who also f o u n d a similar effect with p e n t o s a n polysulfate, a semisynthetic sulfated polysaccharide. S m o o t h muscle cells in p r i m a r y culture can also be m a i n t a i n e d with a high Vvmyo by seeding the freshly dispersed cells at confluent density [15] or by placing sparsely seeded cells with a spatially s e p a r a t e d feeder layer of confluent high V~myo s m o o t h muscle cells or confluent endothelial cells [22]. B o t h cell types are k n o w n to p r o d u c e large a m o u n t s of an antiproliferative h e p a r a n sulfate species which is readily i n c o r p o r a t e d into their own basal lamina [23]. It t h u s appears t h a t the presence of heparinlike g l y c o s a m i n o g l y c a n s is an i m p o r t a n t d e t e r m i n a n t of the p h e n o t y p e t h a t s m o o t h muscle expresses a n d t h a t high levels of this g l y c o s a m i n o g l y c a n species m a i n t a i n the cells in a high Vvmyo state. A n y factor which removes this s u b s t a n c e (e.g., e n z y m e dispersion, w a s h i n g a n d dilution t h r o u g h sparse-seeding) m a y t h e r e f o r e induce the cells to u n d e r g o a c h a n g e in p h e n o t y p e to a low Vvmyo state. S m o o t h muscle cells which have been seeded sparsely o n t o a layer of type IV collagen or basem e n t m e m b r a n e Matrigel (a solubilized e x t r a c t of E H S t u m o r b a s e m e n t m e m b r a n e c o n t a i n i n g collagen type IV, laminin, h e p a r a n sulfate proteoglycan, a n d entactin) do not u n d e r g o a change in p h e n o t y p e , nor do isolated s m o o t h muscle cells which have been c o m p l e t e l y e m b e d d e d in a gel of collagen type I [24]. T h u s replacem e n t of certain c o m p o n e n t s of the basal lamina, or providing a m i c r o e n v i r o n m e n t in which the basal l a m i n a can be rapidly reconstituted, e n c o u r a g e s m a i n t e n a n c e of the high VvmyO state. P e r i t o n e a l m a c r o p h a g e s grown in coculture with a confluent m o n o l a y e r of high V~myo s m o o t h muscle cells induce a significant decrease in Vvmyo after 3 days as c o m p a r e d with b o t h the freshly isolated s m o o t h muscle cells a n d those grown for 3 days in the absence of m a c r o p h a g e s [25]. Since invasion of the a r t e r y wall by m o n o cytes (which s u b s e q u e n t l y b e c o m e m a c r o p h a g e s ) is one of the earliest cellular events in e x p e r i m e n t a l atherogenesis [26], it m a y be t h r o u g h the influence of these cells t h a t s m o o t h muscle p h e n o t y p i c c h a n g e is induced in the initial stages of the disease. In the p r e s e n t study, an assay previously described by Savion et al. [27] is used to d e m o n s t r a t e t h a t m a c r o p h a g e s completely degrade h e p a r a n sulfate-rich m a t r i x

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to low-molecular-weight f r a g m e n t s a n d t h a t the subcellular site of e n z y m e activity is the lysosome. F u r t h e r more, when a confluent m o n o l a y e r of living s m o o t h muscle cells is labeled with 35S, m a c r o p h a g e s b o t h degrade the labeled h e p a r a n sulfate p r o t e o g l y c a n on the s m o o t h muscle surface a n d induce a c h a n g e in s m o o t h muscle p h e n o t y p i c expression, indicating t h a t r e m o v a l of h e p a r a n sulfate from the pericellular c o m p a r t m e n t o f s m o o t h muscle cells m a y be the trigger t h a t initiates s m o o t h muscle p h e n o t y p i c change. MATERIALS AND METHODS Smooth muscle cell culture. The thoracic and abdominal aortic media from 9- to 12-week-old rabbits was dispersed into single smooth muscle cells with collagenase and elastase [15] and seeded into 30-ram culture dishes (Sterilin) at 8 • 105 cells/dish (i.e., 1.1 • 105 cells/cm 2) in medium 199 (M199) plus 5% fetal calf serum (FCS) and 2 mM glutamine. At this seeding density the cells were confluent upon attachment and flattening after 24 h. The culture medium was changed on Days 3 and 5. On Day 8, the medium was again changed and 100 #l of macrophage lysosomal lysate, 10 units/ml heparinase from Flavobacterium heparinum (Sigma Chemical Co., St. Louis), 0.001 units/ml heparitinase (Seikagaku Kogyo Co., Tokyo), 10 #g/ml trypsin, collagenase, or elastase (Sigma), or 10 units/ml chondroitin ABC lyase (Sigma) was added and the cultures were maintained for a further 4 days. Control smooth muscle cultures contained no additives. Ultrastructural morphometry. The smooth muscle cell cultures were fixed and processed for electronmicroscopy (see [15]). After determination of the number of photographs required per dish to give a statistical variation within 5%, between 90 and 135 photographs per experimental group were morphometrically analyzed by point counting to determine the Vvmyo(see [1]). Three blocks were analyzed per dish, and there were three dishes per treatment. R N A extraction and Northern blot hybridization. Cultured cells were scraped from Petri dishes and homogenized in 4.5 M guanidinium thiocyanate, 50 mM EDTA, 25 mM sodium citrate, 0.1 M 2-~-mercaptoethanol, and 2% laurylsarcosine and RNA was prepared [15]. Total RNA (2 #g/lane) was denatured with glyoxal and electrophoresed in 1% agarose gels in 10 mM phosphate buffer, pH 6.8. The RNAs were then transferred to Biodyne filters (Pall Ultrafine Filtration Corp., Glencoe, NY), prehybridized, then hybridized with SP6RNA polymerase transcribed ~2P-labeled cRNA probes. The probes used were derived from the coding region [total actin probe (pRAo A-C): a 320-bp BglII-AvaII fragment corresponding to the sequence coding for the amino acids 185 to 291] and from 3' untranslated region of the a-smooth muscle actin mRNA [a-smooth muscle probe (pRAo a A-3ffJT): a 130-bp DdeI-HindIII fragment] [28]. The fraction of a-smooth muscle actin mRNA as a percentage of total actin mRNA was obtained by calculating the ratio between the 1.7-kbband and the sum of the 1.7- and 2.1-kb bands by means of computerized densitometric scanning [15]. Macrophage culture. Macrophages were isolated from the peritoneal cavity of Balb/c mice and 12-week-old rabbits by lavage [25]. The average yield was l0 s per animal (both mouse and rabbit), cellular purity >90% as judged by erythrophagocytosis, and survival time in culture about 7 days. Because of the relative low yield from rabbits, we used mouse peritoneal macrophages for most experiments. However, all critical experiments were done at least once with rabbit macrophages and the results found to be identical with those obtained with mouse macrophages. Furthermore, for some experiments the J774 cell line of mouse peritoneal macrophages [25] was used, which

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produced identical results to rabbit and mouse peritoneal macrophages. All cells were grown in M199 + 5% FCS + glutamine (2 raM). To prepare macrophage-conditioned medium , 90-mm culture dishes each containing 1.8 • 107 mouse peritoneal macrophages or J774 macrophages were incubated at 37~ for 24 h in 5 ml M199 minus serum (M199-S). The s u p e r n a t a n t was centrifuged at 300g and either used immediately or concentrated more t h a n 10-foLd using an Amicon concentrator (Amicon Corp., Danvers, MA). Crude fractions were prepared by adding progressively higher concentrations of ammonium sulfate (after the addition of 1% bovine serum albumin as carrier), and centrifuging and removing each precipitate before adding more ammonium sulfate. Four precipitates were obtained from 0-25%, 25-50%, 50-75%, and 75-100% a m m o n i u m sulfate.

Assay for degradation products of ass-labeled proteoglycans. Freshly dispersed rabbit aortic smooth muscle cells were plated at confluent density (9 • 10~ cells/30-mm dish which is 1.3 • 10~ cells/ cm z) in M199 + 5% FCS + glutamine (2 raM). On Day 3 the medium of the cultures was changed and Na213sS]O4 added to a final concentration of 30 ttCi/ml. On Day 7 additional radiolabel was added without changing or adding medium. The following day cultures were thoroughly washed in Dulbecco's phosphate-buffered saline (DPBS) a n d the cells dissolved with 0.5% Triton X-100 in DPBS followed by exposure to 25 mM NH4OH. The culture dishes were then extensively washed with DPBS. The 35S-labeled extracellular matrix remained intact and firmly attached to the entire bottom of the culture dish and could be stored at 4~ for up to 1 m o n t h before use [27]. T h e initial studies were done using the heparan sulfate-rich matrix laid down by endothelial cells as in the studies by Savion et al. [27]. However, after these pilot studies, all experiments utilized matrices laid down by smooth muscle. The zhS-labeled extracellular matrix was incubated for 24 h at 37~ with 1.5 ml fresh M199-S (pH 7.0) or with mouse peritoneal macrophages or J774 macrophages at 1 • 105 to 1 • 10~ cells/ml in 1.5 ml M199-S, 100 ttl J774 lysosomal lysate or plasma membranes, J774conditioned medium minus serum, or in the presence of heparinase (10 units/ml), heparitinase (0.001 units/ml), chondroitin ABC lyase (10 units/ml), or trypsin, collagenase, or elastase (10 t~g/ml). In some additional experiments, the incubations also included 5% FCS. Some incubations were repeated in the presence of 10 #g/ml sodium heparin (Sigma). T h e s u p e r n a t a n t was centrifuged at 150g for 5 rain to remove macrophages (if present) then at 10,000g (5 min), and 0.5-ml aliquots applied for gel filtration on Sepharose 6B columns (0.7 • 35 cm) equilibrated with PBS containing 0.1% sodium azide. Fractions (180 • 0.2 ml) were collected at a flow rate of 5 m l / h and counted for radioactivity using a Tri-Carb liquid scintillation analyzer {Model 3255 CA, P a r k a r d I n s t r u m e n t Co., IL). The excluded or void volume (V0) and total volume (Vt) were marked by passage of Dextran Blue 200 and phenol red respectively. Similar gel filtration profiles (Kay values) for all samples including Dextran Blue and phenol red were obtained whether the centrifuged medium was subjected to gel filtration under dissociation conditions (4 M guanidine hydrochloride in 1 M sodium acetate at pH 5.5) or eluted with PBS [27]. Recovery of labeled material from the columns averaged 90%, and each experiment was performed a minimum of three times with variation in elution profiles (Kay values) of less t h a n 10%.

Labeling of sulfated proteoglycans in the smooth muscle basal lamina. Freshly dispersed rabbit aortic smooth muscle cells were plated at confluent density (see above) in 30-mm dishes. After 7 days in primary culture, Na213~S]O4 was added to a final concentration of 30 #Ci/ml. After 24 h the living cells were thoroughly washed several times with DPBS and t h e n used immediately. Under these conditions it was determined t h a t 87% of the radioactivity was associated with the cell surface (trypsin releasable), 1% inside the cells, and 12% incorporated into the extracellular matrix. Pulse-chase. To examine the fate of heparan sulfate on the surface of smooth muscle cells, the culture monolayer was labeled with 3~S as above, except t h a t the labeling time was only 1 h. Under these

conditions, no radioactivity was present in extracellular matrix, with 99% of the incorporated 35S present on the cell surface (as heparan sulfate proteoglycan) and 1% was inside the cells. After the labeling period the monolayer was thoroughly washed t h e n chased over a 24-h period in the presence of culture medium. T h e amount of radioactivity associated with the'cell surface, inside the cells and released into the culture medium was determined at regular intervals and the form of the labeled molecule determined by passing aliquots of each sample through Sepharose 6B and plotting the elution profile (see above). In some studies, chloroquine (100 raM) or NH4C1 (10 mM) was present in order to inhibit lysosomal enzymes in the smooth muscle cells. Macrophage lysosomal lysate. Fifty 90-mm dishes of confluent J774 macrophages (9 • 108 cells) were scraped into DPBS, then centrifuged at 200g for 5 rain. The cell pellet was resuspended in an ice-cold solution of 250 mM sucrose, 3 mM imidazole, and 0.1% absolute ethanol (SIE) at pH 7.4. The cells were homogenized and the lysosomes separated by centrifugation through percoll. The percoll was washed away by diluting in sucrose/KH2PO4, centrifuged at 30,000g for 5 min and the resulting pellet resuspended in 1 ml of sucrose/KH2PO4, freeze-thawed several times to lyse the lysosomes, t h e n stored at - 2 0 ~ (see [29]). To check t h a t the pellet consisted of lysosomes, the activity of acid phosphatase was measured spectrofluorometrically, against a standard curve of 10-1000 ng/ml 4-methylumbelliferone (see [17]). The degree of contamination with plasma membranes was determined by assay of 5'-AMPase activity [30]. Macrophage plasma membranes. Confluent 90-mm dishes of J774 macrophages were scraped into 1 m M N a H C O J 0 . 5 m M CaCl 2. The cells were centrifuged at 200g for 5 min, and the pellet was resuspended in N a H C O J C a C I 2 and homogenized in a loose-fitting Dounce homogenizer. The washed homogenate was t h e n resuspended in 72% (w/v) sucrose to give a final concentration of 62% (w/v) and overlayered with 54, 48, 43, and 8% (w/v) sucrose layers. After centrifugation at 98,000g for 2 h, the plasma membrane faction was collected and assayed for 5'-AMPase and acid phosphatase activity [31]. Statistical analysis. To compare treated and control samples in relation to Vvmyo or a-actin mRNA as a percentage of total actin mRNA, Student's t test was performed.

RESULTS

Ultrastructural M o r p h o m e t r y J 7 7 4 lysosomal lysate. I t h a s p r e v i o u s l y b e e n r e ported from this laboratory that in the presence of mouse peritoneal macrophages, the Vvmyo of confluent smooth muscle cells from the rabbit aorta was signific a n t l y r e d u c e d a f t e r 3 d a y s i n p r i m a r y c u l t u r e [25]. T o determine whether the lysosomes of macrophages cont a i n e d t h e s u b s t a n c e r e s p o n s i b l e f o r t h i s e f f e c t , 1 0 0 #1 J774 macrophage lysosomal lysate was added to confluent 30-mm dishes of rabbit aortic smooth muscle, 8 days in culture, which had a Vvmyo of 49.3 + 0.4%. These dishes had been seeded at near-confluency on D a y 0 a n d c o n t a i n e d 1.5 m l f r e s h M 1 9 9 w i t h 5 % F C S and 2 mM glutamine. The lysosomal fraction had a high activity of acid phosphatase and negligible 5'-AMPase activity showing that there was minimal contamination with plasma membranes. After 4 days in the presence of the lysosomal lysate, the Vvmyo of the cells was 38.2 + 2.6%, which was signific a n t l y l o w e r ( p < 0.1) t h a n t h a t o f c o n t r o l c e l l s ( 4 8 . 2 _+

MODULATION

OF SMOOTH

0.5%). Th er e also was a significant difference (p < 0.05) in the level of a-actin mRNA, as a percentage of total actin mRNA, between control cells (88.9 + 1.3%) and those in the presence of the lysosomal lysate (70.6 + 2.7%). By this we do not imply t hat the actual amount of a-actin mRNA per cell is decreased, merely t hat its level has altered in relation to fl- and ~-actin mRNA. We have previously shown t hat a-actin mRNA as a percentage of total actin mRNA is a reliable marker for smooth muscle phenotype [15]. Macrophage-conditioned medium. Medium freshly conditioned by confluent mouse peritoneal macrophages (4 • 10s cells/ml M199-S for 24 h at 37~ was mixed with an equal volume of M199 containing 10% FCS (to give a final concentration of 5% FCS) and added to confluent 30-mm dishes of rabbit aortic smooth muscle, 8 days in primary culture. After 4 days, the Vvmyo of the smooth muscle cells was 47.3 _+ 0.4%, which was not significantly different from t h a t of control cells (49.2 _+ 1.4%). Crude fractions were obtained from a 10-fold concentrate of macrophage-conditioned medium by progressive addition of 0-25, 25-50, 50-75, and 75-100% ammonium sulfate. Each of the four precipitates was dissolved in 1 ml M199-S, and 100 #l aliquots were added to confluent 30-mm dishes of rabbit aortic smooth muscle in fresh M199 + 5% FCS + 2 m M glutamine. After 4 days, the Vvmyo of smooth muscle cells in the presence of fractions formed by 0-25, 25-50, and 75-100% ammonium sulfate were not significantly different from controls (p > 0.5), but the Vvmyo of smooth muscle cells in the presence of fraction 50-75% had a Vvmyo of 42.7 + 2.9%, which was lower than t hat of control cells (49.2 _+ 1.4%, p < 0.1). This fraction also was shown to have heparan sulfate-degrading activity while the other fractions did not (see later). Aliquots (100 pl) of each of the four fractions were also added to cultures of passage 1 smooth muscle cells seeded below confluency at 2 • 105 cells/dish (3 • 104 cells/cm2). Cells in the presence of fraction 25-50% were stimulated to proliferate above the level of controls while there was no effect with any of the other fractions (see Fig. 1). Since the mitogenic activity and the substance inducing a decrease in Vvmyo of smooth muscle appeared in different fractions, it was clear t h a t these two factors were quite separate. Heparinase/heparitinase. Similarly, to confluent aortic smooth muscle cells 8 days in primary culture (VvmyO = 49.3 + 0.4%) was added 10 units/ml heparinase or 0.001 units/ml heparitinase in 1.5 ml fresh culture medium. After a further 4 days, cultures were fixed in 5% glutaraldehyde and processed for electron microscopy. Th e VvmyO of control cells was found to be 51.0 _+ 3.2%, while th at of heparinase- or heparitinase-treated cells was lower (p < 0.1) at 42.8 _+ 1.2 and 44.6 _+ 1.8%, respectively. Higher concentrations (50 and 100 uni t s/

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DAYS IN CULTURE

F I G . 1. G r o w t h c u r v e s of p a s s a g e 1 s m o o t h m u s c l e cells f r o m t h e rabbit a o r t a s e e d e d below c o n f l u e n c y at 2 • 105 c e l l s / 3 0 - m m d i s h in t h e p r e s e n c e of s e q u e n t i a l (NH4)2SO4-precipitated f r a c t i o n s f r o m a c o n c e n t r a t e of m a c r o p h a g e - " c o n d i t i o n e d " m e d i u m . T h e fraction precipitated by 2 5 - 5 0 % (NH4)2SO 4 (- - - ) s t i m u l a t e d t h e proliferation o f s m o o t h m u s c l e cells above t h a t of c o n t r o l s ( - - ) , w h e r e a s f r a c t i o n s p r e c i p i t a t e d by 0 - 2 5 % ( - . -), 5 0 - 7 5 % (. 9 9 ), a n d 75-100% ( - - - ) h a d no effect.

ml heparinase and 0.01, 0.05, and 0.1 units/ml heparitinase) produced similar results, while lower concentrations (1 unit/ml heparinase and 0.0001 units/ml heparitinase) had no effect. T he levels of a-actin mRNA were significantly lower (p < 0.05) in 10 units/ml heparinase and 0.001 units/ml heparitinase-treated cells (72.4 + 3.1 and 70.6 _+ 4.1%) compared with controls (90.6 + 1.1%). No change in Vvmyo or a-actin m RN A levels were observed when 10 #g/ml sodium heparin was included in the incubation (48.6 + 1.2 versus 51.0 + 3.2% control and 88.2 _+ 2.4 versus 90.6 _ 1.1% control, respectively). Trypsin/chondroitinase. T o determine whether the effect on smooth muscle phenotype was specific for heparan sulfate/heparin-degrading enzymes, 8-day confluent cultures of rabbit aortic smooth muscle were treated with 10 ttg/ml trypsin or 10 units/ml chondroitin ABC lyase for 4 days. In both cases, there was no significant difference (p > 0.5) in Vvmyo (51.2 _ 2.1 versus 51.0 _+ 3.2%) or ~-actin mRNA (90.2 _+3.0 versus 90.6 _ 1.1%) levels between treated and control dishes. Higher concentrations of chondroitin ABC lyase (50 and 100 units/ml) were also ineffective. Higher concentrations of trypsin were not tested for fear of damage to the cell membrane with nonspecific changes over a 4day incubation.

Degradation of Sulfated Proteoglycans T h e 35S-labeled smooth muscle-derived extracellular matrix was completely solubilized with pronase, and 0.3-ml aliquots of the digest were t reat ed with either chondroitin ABC lyase or nitrous acid. Glycosaminogly-

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cans resistant to each degradation procedure were reisolated by precipitation with 1% cetylpyridium chloride and dissolved in 0.3 ml water, and their radioactivity was measured. By also measuring the radioactivity of the supernatant and of the untreated digest of solubilized matrix, the level of heparan sulfate and chondroitin sulfate as a percentage of total sulfated glycosaminoglycan was calculated to be approximately 70% heparan sulfate, 20% chondroitin sulfate, and 10% other, in agreement with published values [32]. M199-S or M199 + 5% FCS (1.5 ml, pH 7.0) was added to the thoroughly washed 35S-labeled extracellular matrix and incubated at 37~ at 24 h. After gel filtration of the supernatant through Sepharose 6B all the radioactivity comigrated with Dextran Blue (Mr 2 X 106 Da), that is, eluted at the void volume, indicating that only high-molecular-weight nondegraded proteoglycans had been released into the medium. Free asSO~- applied directly to the column eluted with a Ka~ of 0.86. After incubation of the matrix with living mouse peritoneal macrophages or J774 macrophages (1 • 106 cells/ml), the peak at Vo was small, and instead more than 80% of the radioactivity eluted with a Kav of 0.84 (Fig. 2), indicating that most of the 35S-labeled glycosaminoglycan chains of the proteoglycans had been completely degraded. When 10 #g/ml sodium heparin was added to the incubation with the living macrophages, the size of the Kav 0.84 peak was decreased, the peak at Vo increased, and a number of small peaks appeared at K~v

0.5 or less (Fig. 2), indicating that proteolytic activity of the macrophages was not inhibited and that large proteoglycans were released into the supernatant but were not degraded further. When 1.8 X 107 J774 macrophages or mouse peritoneal macrophages were lysed with 0.05% Triton X-100 in 3 ml phosphate-buffered saline and a 100-#l aliquot was incubated in 1.4 ml M199-S or M199 + 5% FCS with 35S-labeled matrix for 24 h at 37~ most of the degradation products eluted with a Kay of 0.60, with smaller peaks at V0, and at Kay 0.84 (Fig. 3). On the basis of a comparison of the experimental K,v values on Sepharose 6B columns with those determined for chondroitin sulfate fractions of known molecular weight [33], it was estimated that the degradation fragments at Kav 0.60 had a molecular weight of about 10,000 Da. Fresh macrophage-conditioned medium (4 • 106 cells/ml M199-S for 24 h at 37~ incubated with 35S-labeled matrix for 24 h resulted in most of the released radioactivity eluting at V0, similar to that observed with fresh M199-S, with minor peaks at K,v 0.60 and 0.84 (Fig. 3). However, when each of the four ammonium sulfate precipitates, obtained after addition of increasing amounts of ammonium sulfate to a concentrate of macrophage-conditioned medium, was dissolved in 1 ml M199-S and 100 ~l aliquots were tested for matrix-degrading activity, the 0-25, 25-50, and 75-100% fractions all produced large peaks at V0 and another broad peak with a K ~ of 0.370.42 (Mr 30,000-40,000 Da), while fraction 50-75% had a minor peak at V0 and a distinct peak at K,v 0.65 (Fig. 4). The matrix-degrading activity of the macrophages was stored in the lysosomes since inhibition of lyso0 I

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somal enzymes with 100 # g / m l chloroquine or 10 mM NH4C1 completely inhibited the degradation of the sulfated glycosaminoglycans b e y o n d large proteoglycans. F u r t h e r m o r e , in the p r e s e n c e of 100 #1 J774 m a c r o p h a g e lysosomal lysate at n e u t r a l pH, a large degradation p e a k with K ~ 0.63 occurred which was totally inhibited w h e n 10 #g/ml h e p a r i n was included in the i n c u b a t i o n (Fig. 5A). W h e n the lysosomal lysate was i n c u b a t e d with 35Slabeled m a t r i x at p H 6.5 or less, the p e a k at K ~ 0.63 was smaller and an additional large peak at K~v 0.84 app e a r e d (Fig. 6). H a v i n g established t h a t the K~v 0.63 peak eluted in fractions 100 to 125, a second aliquot of the lysate was run on the same c o l u m n and fractions 100 to 125 were collected (5 ml). An aliquot (0.5 ml) of these pooled fractions was t r e a t e d with a m i x t u r e of 50 #l of 2.4 M NaNO2 and 50 ttl glacial acetic acid (0.2 M nitrous acid) for 80 min at 25~ followed by 100 #1 2 MNa2CO~ to stop the reaction. T h i s low p H nitrous acid t r e a t m e n t selectively results in d e a m i n a t i o n of h e p a r a n sulfate chains to f r a g m e n t s less t h a n M, 1,000 [34]. Indeed, w h e n this mixture was passed t h r o u g h S e p h a r o s e 6B, no peak at K~v 0.63 occurred, b u t instead a peak at K ~ 0.84 a p p e a r e d (Fig. 5B). In contrast, t r e a t m e n t of a n o t h e r 0.5-ml aliquot of pooled fractions 100-125 (peak K~v 0.63) with 10 u n i t s / m l c h o n d r o i t i n ABC lyase, which degrades chondroitin sulfate chains, resulted in only a very small peak at K ~ 0.84, with most of the radioactivity continuing to elute with a Kav 0.63 (Fig. 5B). Addition o f 10 u n i t s / m l h e p a r i n a s e at p H 7.0 to ma-

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162

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Degradation of Smooth Muscle Basal Lamina and Change in Phenotype In order to d e m o n s t r a t e a direct correlation b e t w e e n degradation of s m o o t h muscle h e p a r a n sulfate a n d a

change in s m o o t h muscle p h e n o t y p e it was n e c e s s a r y to add living m a c r o p h a g e s to living s m o o t h muscle cells with 35S-labeled h e p a r a n sulfate in t h e i r pericellular c o m p a r t m e n t . T h e pericellular c o m p a r t m e n t of confluent, high VvmyO s m o o t h muscle cells 7 days in culture was radiolabeled over a 24-h period. U n d e r these conditions it was f o u n d t h a t 87% of the i n c o r p o r a t e d 35S was associated with the cell surface, 12% with the deposited extracellular matrix, a n d 1% was inside the cells. Using sister dishes c o n t a i n i n g the same n u m b e r of radiolabeled s m o o t h muscle cells, different n u m b e r s of rabbit m a c r o p h a g e s over a 100-fold range were added for 24 h a n d the elution profile of degradation p r o d u c t s was examined. T h e area u n d e r the peak at Kay 0.84 increased with increasing n u m b e r s of m a c r o p h a g e s until a p l a t e a u was r e a c h e d a n d the size of the peak did not increase further. S u b s e q u e n t h e p a r i t i n a s e t r e a t m e n t of these cells showed t h a t no labeled h e p a r a n sulfate was remaining on the cell surface. B y plotting the i n t e g r a t e d area u n d e r the p e a k against the n u m b e r of m a c r o p h a g e s a n d knowing the n u m b e r of s m o o t h muscle cells p r e s e n t in the monolayer, it was calculated t h a t one m a c r o p h a g e could remove all the h e p a r a n sulfate f r o m the surface of t h r e e s m o o t h muscle cells (Fig. 8A). T h i s was accompanied (in cultures where the ratio of m a c r o p h a g e s to s m o o t h muscle was 1:3) by a decrease in t h e Vvmyo of the same s m o o t h muscle cells to 32.1% after 24 h comp a r e d with control s m o o t h muscle cells with no macrophages whose Vvmyo was 51%, and a significant decrease in a-actin m R N A as a p e r c e n t a g e of total actin

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MODULATION OF SMOOTH MUSCLE PHENOTYPE MACROPHAGES : SMC 1:9

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m R N A (70.1 _+ 2.6 v e r s u s 90.8 • 1.7%). F u r t h e r m o r e , a f t e r 3 days the n u m b e r of s m o o t h muscle cells in t h e cultures i n c r e a s e d significantly w h e n t h e ratio of m a c r o p h a g e s to s m o o t h muscle was 1:3 a n d above, e v e n t h o u g h the s m o o t h muscle cells were a l r e a d y c o n f l u e n t (Fig. 8B). T h i s indicated t h a t the m e c h a n i s m b y which m a c r o p h a g e s induce a change in s m o o t h muscle p h e n o t y p e is s o m e h o w r e l a t e d to t h e i r d e s t r u c t i o n of all hepa r a n sulfate f r o m the s m o o t h muscle cell surface, a n d t h a t following p h e n o t y p i c c h a n g e t h e s m o o t h muscle cells r e s p o n d e d to m i t o g e n s released b y the m a c r o p h a g e s a n d / o r b y the p h e n o t y p i c a l l y a l t e r e d s m o o t h muscle cells t h e m s e l v e s .

163

Since in the a b s e n c e of m a c r o p h a g e s t h e r e was s o m e d e g r a d a t i o n of t h e h e p a r a n sulfate p r o t e o g l y c a n b y t h e s m o o t h muscle cells t h e m s e l v e s , s o m e cultures were airdried, killing the s m o o t h muscle cells, b u t m a i n t a i n i n g an i n t a c t 35S-labeled b a s a l lamina. T h e s t u d y was t h e n r e p e a t e d by adding different n u m b e r s of m a c r o p h a g e s to t h e s a m e n u m b e r of c o n f l u e n t s m o o t h m u s c l e cells a n d s u b s t a n t i a l l y t h e s a m e result was obtained. T h a t is, one m a c r o p h a g e d e g r a d e d all t h e r e m o v a b l e h e p a r a n sulfate f r o m t h r e e s m o o t h m u s c l e cells. T h e fact t h a t t h e c o n t r a c t i l e s m o o t h muscle cells were internalizing a n d d e g r a d i n g t h e i r own h e p a r a n sulfate was itself intriguing, as it suggested t h e m e c h a n i s m b y which t h e h e p a r a n sulfate in t h e b a s a l l a m i n a m a i n t a i n e d t h e cells in a high Vvmyo. In o t h e r cell s y s t e m s it h a s b e e n s h o w n t h a t cells regularly internalize h e p a r a n sulfate on t h e i r surface a n d d e g r a d e it, b u t t h a t c e r t a i n f r a c t i o n s of h e p a r a n sulfate e n r i c h e d in t h e r a r e 2 - 0 sulfate g l u c u r o n a t e u n i t s are n o t fully d e g r a d e d b u t are t r a n s p o r t e d to t h e nucleus w h e r e t h e y influence gene e x p r e s s i o n [35]. T o e x a m i n e t h e i n t e r n a l i z a t i o n a n d degr a d a t i o n of h e p a r a n sulfate b y s m o o t h muscle cells with a high Vvmyo, t h e living, c o n f l u e n t cells were pulse labeled with 35S for 1 h only, t h e n t h e a m o u n t of radioactivity a n d its elution profile released (i) into the culture m e d i u m , (ii) on t h e surface of t h e cell ( t r y p s i n releasable), a n d (iii) within the cell (trypsin n o n r e l e a s a b l e ) m e a s u r e d o v e r a 24-h c h a s e period. An aliquot of e a c h s a m p l e was also p r e t r e a t e d with h e p a r i t i n a s e before gel filtration in order to d e t e r m i n e the relative c o n t r i b u t i o n b y h e p a r a n sulfate. T h e a m o u n t of [85S]heparan sulfate p r o t e o g l y c a n on the cell surface d e c r e a s e d e x p o n e n tially with time, b u t a f t e r 24 h, a b o u t a t h i r d was still r e m a i n i n g (Fig. 9). N o i n t a c t h e p a r a n sulfate p r o t e o g l y c a n (Kay < 0.32) was r e l e a s e d into t h e i n c u b a t i o n m e d i u m at a n y t i m e d u r i n g the 24-h chase, a n d indeed only totally d e g r a d e d h e p a r a n sulfate (Ka~ > 0.83) was found. W i t h i n the s m o o t h m u s c l e cells, a s u b s t a n t i a l a m o u n t of i n t a c t h e p a r a n sulfate p r o t e o g l y c a n (Ka~ < 0.32) was p r e s e n t i m m e d i a t e l y at the e n d of t h e p u l s e - l a b e l i n g period a n d o v e r t h e n e x t 6 h, p e a k s at 0.70 > Ka~ > 0.32 a p p e a r e d . Only small p e a k s at K ~ > 0.70 o c c u r r e d within t h e cells, indicating t h a t as soon as t h e h e p a r a n sulfate was c o m p l e t e l y d e g r a d e d m o s t of it rapidly left t h e cell. T h e t i m e course a n d size o f the p e a k s were o n l y slightly d e p r e s s e d in the p r e s e n c e of either NH4C1 or chloroquine, indicating t h a t m o s t of t h e d e g r a d a t i o n of h e p a r a n sulfate in s m o o t h muscle cells o c c u r r e d via a n o n l y s o s o m a l p a t h w a y . W i t h m a c r o p h a g e s , NH4C1 or c h l o r o q u i n e c o m p l e t e l y inhibited d e g r a d a t i o n of h e p a r a n sulfate p r o t e o g l y c a n , indicating t h a t t h e m a c r o p h a g e p a t h w a y was via lysosomes. Finally, in o r d e r to d e t e r m i n e w h e t h e r s m o o t h muscle cells of different p h e n o t y p e h a d different capacities for d e g r a d a t i o n of h e p a r a n sulfate, t h e s a m e n u m b e r of s m o o t h muscle cells of different p h e n o t y p e were soni-

164

CAMPBELL E T AL. SMC BASAL LAMINA

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