AJH
1991;
4:183-188
Timothy Scott-Burden, Thérèse J. Resink, Alfred W. A. Hahn, and Fritz R. Bühler
S m o o t h m u s c l e cells from s p o n t a n e o u s l y h y p e r t e n sive r a t s (SHR) p r o l i f e r a t e i n c u l t u r e faster t h a n t h o s e isolated from sex- a n d a g e - m a t c h e d W i s t a r Kyoto (WKY) a n i m a l s . T h e r e w a s n o difference i n t h e k i n e t i c s of S6 k i n a s e a c t i v a t i o n i n t h e t w o cult u r e s , b u t later m e t a b o l i c e v e n t s associated w i t h p r o l i f e r a t i o n w e r e s t i m u l a t e d e a r l i e r i n S H R cells t h a n i n WKY, eg, a c t i v a t i o n of o r n i t h i n e d e c a r b o x ylase. Both cell t y p e s e l a b o r a t e d a n e x t e n s i v e extracellular m a t r i x i n c u l t u r e c o m p o s e d of a different
b l e n d of c o n n e c t i v e t i s s u e m a c r o m o l e c u l e s . M a t r i x m a t e r i a l from SHR cells w a s m o r e s t i m u l a t o r y to g r o w t h of WKY c u l t u r e s t h a n t h e i r o w n m a t r i c e s . Angiotensin stimulated the growth and synthesis of e x t r a - c e l l u l a r m a t r i x m a t e r i a l i n S H R m o r e t h a n i n WKY d e r i v e d v a s c u l a r s m o o t h m u s c l e cell c u l t u r e s . A m J H y p e r t e n s 1991;4:183-188
T
elude c o m p o u n d s that h a v e b e e n characterized as g r o w t h factors, as well as a n u m b e r of molecules normally associated with the structural c o m p o n e n t s of the vessel wall (see Figure 1). A m o n g these extracellular matrix c o m p o u n d s , t h r o m b o s p o n d i n (TS) h a s certain attributes w h i c h strongly suggest that it could play a n important role in a variety of processes that eventually lead to the d e v e l o p m e n t of vascular d i s e a s e . The vasoactive peptide h o r m o n e s are a n o t h e r group of molecules that h a v e received attention w i t h regard to their pathogenic potential. In this respect angiotensin II (Ang II) h a s been suggested to induce irreversible vascular changes. Clearly, the latter is a candidate eminently suited to playing a role in the chronic progressive induction of the vascular structural changes that are part a n d parcel of essential hypertension (EHT), since m o r e t h a n t w o thirds of patients with EHT exhibit elevated plasma r e n i n / A n g II levels relative to the height of their blood pressure. Recent reports h a v e s h o w n that indeed A n g II is capable of stimulating a n u m b e r of metabolic events that m a y lead to s m o o t h muscle proliferative
h e well established concepts embodied in the "response to injury theory," as proposed by Ross a n d Glomset, has provided a model for the study of the m a n y factors a n d their interaction responsible for the pathogenesis of vascular disease u n d e r situations w h e r e frank d e n u d a t i o n of the e n d o thelium h a s taken p l a c e . There is n o w evidence to suggest that both h y p e r t r o p h y a n d hyperplasia can occur in the absence of any a p p a r e n t d a m a g e to the endothelial l a y e r . For these processes to take place w e n e e d to invoke the influences of c o m p o u n d s that are normally present in the intact blood vessel wall. Candidates for the processes m e n t i o n e d abo "* in1
2 - 4
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From the Department of Research, University Hospital, Basel, Switzerland. This study w a s supported by the Swiss National Fund Number 3.817.087. Address correspondence and reprint requests to Professor F. R. Bühler, Department of Research, University Hospital, Ch-4031 Basel, Switzerland.
© 1991 by the American Journal of Hypertension,
Inc.
KEY W O R D S : A n g i o t e n s i n , v a s c u l a r s m o o t h m u s c l e growth metabolism.
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Angiotensin-Induced Growth Related Metabolism Is Activated in Cultured Smooth Muscle Cells From Spontaneously Hypertensive Rats and Wistar-Kyoto Rats
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1991-VOL
MATERIALS A N D
4, NO. 2, PART 1
METHODS
All materials a n d media for tissue culture were obtained from sources already described. The radioisotopes used in the studies described herein were p u r c h a s e d from A m e r s h a m Radiochemical, A m e r s h a m , Bucking h a m s h i r e , England. Immunologic reagents were o b tained from Dakopatts, Glostrup, Denmark. SHR a n d WKY rats were supplied by Madörin/BRL, Frenkendorf, Switzerland. All cell culture techniques a n d details of procedures employed for the isolation of vascular s m o o t h muscle cells (VSMC) from rats (SHR a n d WKY) h a v e b e e n re ported before. Cell n u m b e r s (growth kinetics) were d e termined o n cell suspensions following enzymatic dis aggregation of cell layers a n d counting in a Coulter (Hialeah, FL) c o u n t e r . Plasma-derived serum (PDS) w a s p r e p a r e d by t h e m e t h o d of Ross a n d Kariya a n d each batch w a s as sessed after sterilization by filtration for its mitogenic potential. Samples (10% final concentration) that stimu lated the incorporation of [ H]-thymidine into D N A to levels greater t h a n 5 % of that obtained using 1 0 % w h o l e blood serum were rejected; P D S w a s used in experi m e n t s at a final concentration of 1%. Preparation of extracellular matrix (ECM)-coated plastic culture vessels a n d dishes, as well as all proce dures for S6 kinase activation of quiescent cultures, were described. The incorporation of [ H]-thymidine into D N A w a s m e a s u r e d as described b e f o r e a n d the kinetics of [ H]glycine incorporation into ECM protein w a s performed as described by ourselves a n d o t h e r s . ' Assays for the activation of ornithine decarboxylase (ODC) w e r e carried out as reported by H o vis et a l . 18
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b e h a v i o r . - - The induction of c-fos, c-myc, a n d P D G F A chain gene expression in cultures of rat s m o o t h m u s cle cells by A n g I I , ' as well as its ability to stimulate protein s y n t h e s i s , ' strongly suggest that chronic expo sure of vascular s m o o t h muscle to this vasoactive p e p tide m a y h a v e pathologic consequences. We report here on our investigations with A n g II using cultured s m o o t h muscle cells from spontaneously hypertensive rats (SHR) a n d their normotensive WistarKyoto (WKY) litter mates. We also report here on the use of plasma-derived serum (PDS) in conjunction with Ang II for the prolonged propagation of cultured rat s m o o t h muscle cells. O u r rationale for the use of P D S is that u n d e r n o r m a l physiologic conditions medial s m o o t h muscle cells w o u l d not come into contact with serum from w h o l e blood, but are rather " b a t h e d by a filtrate of p l a s m a , " essentially similar to the n o n mitogenic preparations w e h a v e used in s o m e of these studies. 11
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RESULTS W h e n quiescent VSMC cultures (SHR a n d WKY) w e r e exposed to 1 0 % fetal calf serum (FCS) there w a s a dif ferential t i m e - d e p e n d e n t activation of a n u m b e r of im portant g r o w t h associated metabolic events w h i c h in cluded S6 kinase, ornithine decarboxylase, D N A synthesis, a n d [ H]-thymidine incorporation (Figure 2). The temporal relationship b e t w e e n these events h a s b e e n well d o c u m e n t e d a n d the time intervals observed b e t w e e n the induction of, for example S6 ki n a s e a n d O D C reflect the proliferation rates of t h e stimu lated cultures. SHR-derived cells exhibited shorter time intervals b e t w e e n the induction of S6 kinase a n d O D C t h a n cells from WKY sources (Figure 2). A l t h o u g h w e h a v e s h o w n previously that A n g II differentially stimu lated S6 kinase activation in quiescent cultures of VSMC from SHR a n d WKY s o u r c e s , w e did not observe a n y induction of O D C activity or stimulation of [ H]-thymi3
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FIGURE 1. Schematic representation of vessel wall and some components thereof which may contribute to the pathogenesis of vascular disease. Matrix molecules: fibronectin (FN); laminin (LN); thrombospondin (TS); tenascin (TN); collagen (COL); heparan sulfate/heparin and elastin (HS). Growth Factors/Stimulators: epidermal growth factor (EGF); basic fibroblast growth factor (FGF ); platelet derived growth factor (PDGF); transforming growth factor β (TGF ); angiotensin (All) and low density lipopro tein (LDL). Furthermore, within the matrix compounds such as LDL and basic fibroblast growth factor can be bound. In the course of the normal turnover of matrix molecules which accompanies the dynamic process of tissue remodelling, peptide fragments having EGF domains can be generated and some of the stimulatory proper ties of this growth factor may become manifest. The compounds known to possess such EGF domains include LN, TS, and TN, a recently discovered matrix glycoprotein also found in vascular tissue. Both endothelial and smooth muscle cells are capable of secreting a number of growth factors without or with agonist stimulation, eg, Ang II. These events may lead to either hyper trophy or hyperplasia as well as the promotion of migration into the intimai space (see Discussion).
AJH-FEBRUARY 1991-VOL 4, NO. 2, PART 1
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Time after FCS addition (hrs) FIGURE 2. Kinetics of activation of important metabolic events leading to cell proliferation in VSMC from SHR and WKY rats. Quiescent cells were exposed to fetal-calf serum (10%) and the activity of S6 kinase, ODC, and the incorporation levels of [ H]thymidine into DNA determined (see Methods). The data repre sents the mean values from two experiments performed on a pair of SHR and WKY isolates at same passage number and at similar cell densities. Closed symbols represent data from SHR and open sym bols, WKY cultures.
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dine incorporation by this agonist w h e n a d d e d to serum-free m e d i u m . The VSMC from SHR a n d WKY sources elaborated extracellular matrices during long-term culture in pres ence of N a ascorbate, w h i c h consisted of a complex blend of connective m a c r o - m o l e c u l e s . Proliferation rates of WKY cells were differentially stimulated w h e n they were cultured u p o n matrices elaborated either by themselves or SHR cells in previous passage (Figure 3). G r o w t h rates of SHR cells were not significantly differ ent w h e n they were plated u p o n matrices elaborated by cells of either type (Figure 3). W h e n quiescent cultures of VSMC from SHR were exposed to A n g II there w a s a t i m e - d e p e n d e n t stimula tion of incorporation of [ C]-glycine a n d [ H]-glucosam i n e into extracellular matrix material above levels ob served for serum-free m e d i u m controls (Figure 4). Similar findings were obtained with cells from n o r m o tensive animals (data not shown). The data also sug gested a differential stimulation of glycoconjugate syn thesis by A n g II since the ratios for the incorporation of the t w o isotopic matrix precursors were significantly (P < .05) different, ( [ H ] / [ C ] ; 1.17 ± 0.2 for A n g II ν 0.77 ± 0 . 2 for controls). Although u n d e r serum-free conditions A n g II w a s incapable of stimulating mitogenic behavior in VSMC, in the presence of 1% P D S cells from SHR exhibited a n e n h a n c e d responsiveness to the agonist leading to a sig27
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FIGURE 3. Growth stimulation by extracellular matrix sub strata. VSMC were plated onto either gelatin-washed or matrixcoated substrata and grown for 3 days with normal medium. Following the growth period wells were trypsinized and cell num bers determined (see Methods). The data is expressed as the per centage of stimulation in growth (cell number) on the different matrix preparations (from SHR or WKY) relative to growth on gelatinized plastic (100°/o) and represents the means ± SD of re peated (n = 4) experiments. The hatched blocks represent data for SHR and * indicates the significance of difference (P > .001) in cell numbers for WKY when plated upon either SHR-derived ECM or their own. 18
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nificant (P < .001) sustained increase in cell n u m b e r (Figure 5). We also observed a minimal increase in prolif eration rates by cells from WKY rats exposed to A n g II a n d 1% P D S , w h i c h only reached significance (P < .01) after 9 days in culture. DISCUSSION VSMC from SHR sources exhibit elevated g r o w t h rates a n d e n h a n c e d responsiveness to g r o w t h stimulants (growth factors, h o r m o n e s , a n d FCS) as c o m p a r e d to cells from WKY s o u r c e s . ' W e h a v e also reported on the differential stimulation of S6 kinase in t h e t w o cell types as a consequence of their exposure to A n g I I . 1 8
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However, this agonist w a s u n a b l e to stimulate t h e acti vation of O D C or subsequent events leading to mitosis u n d e r serum-free conditions. We h a v e also s h o w n that other potential g r o w t h enhancers like low density lipo proteins (LDL) a n d t h r o m b o s p o n d i n (TS) b e h a v e in a similar m a n n e r . ' FCS, w h i c h contains a n u m b e r of mitogens for VSMC (ie, platelet derived g r o w t h factor (PDGF), basic fibroblast g r o w t h factor (FGF ), a n d epi dermal g r o w t h factor (EGF)) stimulated the induction of all three g r o w t h related metabolic events depicted in Figure 2. Cells from SHR r e s p o n d e d faster to FCS in terms of O D C activation a n d [ H]-thymidine incorpora tion t h a n did their normotensive (WKY) counterparts; this is a reflection of their faster g r o w t h rates in culture. Ang II stimulation of matrix glycoconjugate synthesis (Figure 4) m a y contribute to the structural changes that are part a n d parcel of vascular disease. The extracellular matrix plays an important role in the m a i n t e n a n c e of p h e n o t y p e , ' ' a n d our observations that matrix m a t e rial from SHR stimulated the g r o w t h of WKY cells to a greater extent t h a n that elaborated by themselves serves to emphasize the importance of this c o m p o n e n t of the vessel wall. We h a v e s u m m a r i z e d some of the current knowledge a n d some of our o w n findings in regard to the interactions of n o r m a l vessel wall c o m p o n e n t s (Fig ure 1). Central to these observations is the evidence that Ang II is capable of stimulating the expression of a n u m ber of important c o m p o u n d s w h i c h include P D G F A chain, TS, a n d T G F ^ . The induction of P D G F A chain expression in VSMC exposed to A n g II h a s already b e e n r e p o r t e d a n d it m a y account for our observation of g r o w t h stimulation by Ang II of cells from SHR in pres ence of 1% P D S (Figure 5). We h a v e found that cell 20
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isolates from SHR express a n d h a v e receptors for P D G F ^ h o m o d i m e r , w h e r e a s WKY-derived cells only express the g r o w t h factor b u t d o not respond to it, since they lack A-type cell surface r e c e p t o r s . The increase in cell n u m b e r observed in cultures from hypertensive an imals maintained in 1 % P D S m e d i u m alone m a y also be accounted for by their responsiveness to P D G F A-chain h o m o d i m e r . T h e synthesis a n d secretion of the AA homodimeric form of P D G F by arterial s m o o t h muscle cells h a s also b e e n observed by o t h e r s . The combina tion of vessel wall c o m p o n e n t s as depicted in the sche matic (Figure 1) can realistically lead to the initiation of neointimal formation w h i c h typifies vascular pathology in m a n a n d experimental animals. We h a v e found that c o m p o u n d s such as TS, LDL, a n d A n g II are able, in cultured VSMC, of e n h a n c i n g metabolic events nor mally associated with p r o l i f e r a t i o n . ' ' ' ' ' TS alone w a s not mitogenic to VSMC, but, w h e n adminis tered in combination with EGF, its mitogenic potential w a s considerably e n h a n c e d . ' A l t h o u g h there is n o evi dence as yet to suggest the presence of EGF (or its close analogue T G F ) in the normal vessel wall, recent data h a s s h o w n that laminin, w h i c h is certainly present, pos sesses EGF-like sequences within its s t r u c t u r e . It is conceivable that during the course of n o r m a l matrix turnover "EGF-like" molecules m a y arise a n d that these in combination with TS could b e h a v e as a strong mito genic mixture. Additionally, the stimulation of TGF^ ex pression by cultured s m o o t h muscle cells in response to A n g II e x p o s u r e m a y account for the influence of the vasoactive peptide on matrix synthesis, since TGF^ h a s b e e n s h o w n to not only m o d u l a t e the g r o w t h of smooth muscle cells b u t also to e n h a n c e their synthesis of extra34
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FIGURE 4. Kinetics of incorpora tion of radioactive matrix precursors into ECM macromolecules by VSMC from SHR. Quiescent cultures of SHR smooth muscle cells were ex posed to either [ C]-glycine or [ H]-glucosamine in the presence or absence of 100 nmol/L Ang II for the number of times shown. The determi nation of the levels of incorporation was performed as described under Methods and the data (mean values ± SD) is typical of that seen in a number of experiments (n = 4). Closed symbols represent cells maintained in the presence of Ang II and open, in its absence.
AJH-FEBRUARY
1991-VOL
4, NO. 2, PART 1
Α.
J
B.
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Majack RA, Goodman LV, Dixit VM: Cell surface throm bospondin is functionally essential for smooth muscle cell proliferation. J Cell Biol 1988;106:415-422.
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Scott-Burden T, Bühler FR: Regulation of smooth muscle proliferative phenotype by heparinoid-matrix interac tions. Trends Pharmacol Sei 1988;9:94-98.
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Scott-Burden T, Resink TJ, Baur U, et al: Amiloride sensi tive activation of S6 Kinase by angiotensin II in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1988,151:583-589.
J
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FIGURE 5. Growth kinetics of VSMC from SHR and WKY in presence and absence of Ang II. Cells were plated into 12-well multiwell plates (1.25 X 10 cells/well) in normal medium con taining 10% whole blood serum. After 18 h medium was replaced with 1% PDS medium (see Methods) and some wells were further supplemented with Ang II (5X10 mol/L) on a daily basis. Me dium (1% PDS) was changed every 48 h and cell numbers deter mined as described previously. Experiments (n = 3) were per formed on cells from (A) SHR and (B) WKY and the data represent the means ± SD. Statistical significance was analyzed using Stu dent's t test for unpaired data and the levels of significance are indicated on the figure as *, Ρ < .01; **, Ρ < .001. 5
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cellular matrix m a c r o m o l e c u l e s . In conclusion, it w o u l d appear that w e n o longer h a v e to invoke d a m a g e to the e n d o t h e l i u m as the stimulus for the changes that lead to neointimal formation since m a n y c o m p o u n d s such as A n g II, alone or in combination with normal constituents of the vessel wall, are potentially capable of pathogenic modulation of medial s m o o t h muscle cells leading to progressive structural h y p e r t r o p h y , as o b served in vascular tissue from hypertensive animals a n d man.
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Berk BC, Vekstein V, Gordon HM, Tsuda T: Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension 1989;13:305-304.
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Campbell JH, Campbell GR: in Campbell JH, Campbell GR (eds): Vascular Smooth Muscle in Culture, Vol. 1. Boca Raton, Florida, CRC Press, 1987, pp 1 5 - 2 1 .
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Scott-Burden T, Resink TJ, Bürgin M, Bühler FR: Extra cellular matrix: differential influence on growth and bio synthesis patterns of vascular smooth muscle cells from SHR and WKY rats. J Cell Physiol 1990;141:267-274.
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Haudenschild CC, Grunwald J, Chobanian AV: Effects of hypertension on migration and proliferation of smooth muscle in culture. Hypertension 1985;7:(suppl 1)101-104.
Ho vis JG, Stumpo DJ, Halsey DL, Blackshear PJ: Effects of mitogens on ornithine decarboxylase activity and messenger RNA levels in normal and protein kinase C-deficient NIH-3T3 fibroblasts. J Biol Chem 1986; 261:10380-10386.
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S C O T T - B U R D E N ET AL
1991;
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Timothy Scott-Burden, Thérèse J. Resink, Alfred W. A. Hahn, and Fritz R. Bühler
S m o o t h m u s c l e cells from s p o n t a n e o u s l y h y p e r t e n sive r a t s (SHR) p r o l i f e r a t e i n c u l t u r e faster t h a n t h o s e isolated from sex- a n d a g e - m a t c h e d W i s t a r Kyoto (WKY) a n i m a l s . T h e r e w a s n o difference i n t h e k i n e t i c s of S6 k i n a s e a c t i v a t i o n i n t h e t w o cult u r e s , b u t later m e t a b o l i c e v e n t s associated w i t h p r o l i f e r a t i o n w e r e s t i m u l a t e d e a r l i e r i n S H R cells t h a n i n WKY, eg, a c t i v a t i o n of o r n i t h i n e d e c a r b o x ylase. Both cell t y p e s e l a b o r a t e d a n e x t e n s i v e extracellular m a t r i x i n c u l t u r e c o m p o s e d of a different
b l e n d of c o n n e c t i v e t i s s u e m a c r o m o l e c u l e s . M a t r i x m a t e r i a l from SHR cells w a s m o r e s t i m u l a t o r y to g r o w t h of WKY c u l t u r e s t h a n t h e i r o w n m a t r i c e s . Angiotensin stimulated the growth and synthesis of e x t r a - c e l l u l a r m a t r i x m a t e r i a l i n S H R m o r e t h a n i n WKY d e r i v e d v a s c u l a r s m o o t h m u s c l e cell c u l t u r e s . A m J H y p e r t e n s 1991;4:183-188
T
elude c o m p o u n d s that h a v e b e e n characterized as g r o w t h factors, as well as a n u m b e r of molecules normally associated with the structural c o m p o n e n t s of the vessel wall (see Figure 1). A m o n g these extracellular matrix c o m p o u n d s , t h r o m b o s p o n d i n (TS) h a s certain attributes w h i c h strongly suggest that it could play a n important role in a variety of processes that eventually lead to the d e v e l o p m e n t of vascular d i s e a s e . The vasoactive peptide h o r m o n e s are a n o t h e r group of molecules that h a v e received attention w i t h regard to their pathogenic potential. In this respect angiotensin II (Ang II) h a s been suggested to induce irreversible vascular changes. Clearly, the latter is a candidate eminently suited to playing a role in the chronic progressive induction of the vascular structural changes that are part a n d parcel of essential hypertension (EHT), since m o r e t h a n t w o thirds of patients with EHT exhibit elevated plasma r e n i n / A n g II levels relative to the height of their blood pressure. Recent reports h a v e s h o w n that indeed A n g II is capable of stimulating a n u m b e r of metabolic events that m a y lead to s m o o t h muscle proliferative
h e well established concepts embodied in the "response to injury theory," as proposed by Ross a n d Glomset, has provided a model for the study of the m a n y factors a n d their interaction responsible for the pathogenesis of vascular disease u n d e r situations w h e r e frank d e n u d a t i o n of the e n d o thelium h a s taken p l a c e . There is n o w evidence to suggest that both h y p e r t r o p h y a n d hyperplasia can occur in the absence of any a p p a r e n t d a m a g e to the endothelial l a y e r . For these processes to take place w e n e e d to invoke the influences of c o m p o u n d s that are normally present in the intact blood vessel wall. Candidates for the processes m e n t i o n e d abo "* in1
2 - 4
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From the Department of Research, University Hospital, Basel, Switzerland. This study w a s supported by the Swiss National Fund Number 3.817.087. Address correspondence and reprint requests to Professor F. R. Bühler, Department of Research, University Hospital, Ch-4031 Basel, Switzerland.
© 1991 by the American Journal of Hypertension,
Inc.
KEY W O R D S : A n g i o t e n s i n , v a s c u l a r s m o o t h m u s c l e growth metabolism.
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Angiotensin-Induced Growth Related Metabolism Is Activated in Cultured Smooth Muscle Cells From Spontaneously Hypertensive Rats and Wistar-Kyoto Rats
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All materials a n d media for tissue culture were obtained from sources already described. The radioisotopes used in the studies described herein were p u r c h a s e d from A m e r s h a m Radiochemical, A m e r s h a m , Bucking h a m s h i r e , England. Immunologic reagents were o b tained from Dakopatts, Glostrup, Denmark. SHR a n d WKY rats were supplied by Madörin/BRL, Frenkendorf, Switzerland. All cell culture techniques a n d details of procedures employed for the isolation of vascular s m o o t h muscle cells (VSMC) from rats (SHR a n d WKY) h a v e b e e n re ported before. Cell n u m b e r s (growth kinetics) were d e termined o n cell suspensions following enzymatic dis aggregation of cell layers a n d counting in a Coulter (Hialeah, FL) c o u n t e r . Plasma-derived serum (PDS) w a s p r e p a r e d by t h e m e t h o d of Ross a n d Kariya a n d each batch w a s as sessed after sterilization by filtration for its mitogenic potential. Samples (10% final concentration) that stimu lated the incorporation of [ H]-thymidine into D N A to levels greater t h a n 5 % of that obtained using 1 0 % w h o l e blood serum were rejected; P D S w a s used in experi m e n t s at a final concentration of 1%. Preparation of extracellular matrix (ECM)-coated plastic culture vessels a n d dishes, as well as all proce dures for S6 kinase activation of quiescent cultures, were described. The incorporation of [ H]-thymidine into D N A w a s m e a s u r e d as described b e f o r e a n d the kinetics of [ H]glycine incorporation into ECM protein w a s performed as described by ourselves a n d o t h e r s . ' Assays for the activation of ornithine decarboxylase (ODC) w e r e carried out as reported by H o vis et a l . 18
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b e h a v i o r . - - The induction of c-fos, c-myc, a n d P D G F A chain gene expression in cultures of rat s m o o t h m u s cle cells by A n g I I , ' as well as its ability to stimulate protein s y n t h e s i s , ' strongly suggest that chronic expo sure of vascular s m o o t h muscle to this vasoactive p e p tide m a y h a v e pathologic consequences. We report here on our investigations with A n g II using cultured s m o o t h muscle cells from spontaneously hypertensive rats (SHR) a n d their normotensive WistarKyoto (WKY) litter mates. We also report here on the use of plasma-derived serum (PDS) in conjunction with Ang II for the prolonged propagation of cultured rat s m o o t h muscle cells. O u r rationale for the use of P D S is that u n d e r n o r m a l physiologic conditions medial s m o o t h muscle cells w o u l d not come into contact with serum from w h o l e blood, but are rather " b a t h e d by a filtrate of p l a s m a , " essentially similar to the n o n mitogenic preparations w e h a v e used in s o m e of these studies. 11
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RESULTS W h e n quiescent VSMC cultures (SHR a n d WKY) w e r e exposed to 1 0 % fetal calf serum (FCS) there w a s a dif ferential t i m e - d e p e n d e n t activation of a n u m b e r of im portant g r o w t h associated metabolic events w h i c h in cluded S6 kinase, ornithine decarboxylase, D N A synthesis, a n d [ H]-thymidine incorporation (Figure 2). The temporal relationship b e t w e e n these events h a s b e e n well d o c u m e n t e d a n d the time intervals observed b e t w e e n the induction of, for example S6 ki n a s e a n d O D C reflect the proliferation rates of t h e stimu lated cultures. SHR-derived cells exhibited shorter time intervals b e t w e e n the induction of S6 kinase a n d O D C t h a n cells from WKY sources (Figure 2). A l t h o u g h w e h a v e s h o w n previously that A n g II differentially stimu lated S6 kinase activation in quiescent cultures of VSMC from SHR a n d WKY s o u r c e s , w e did not observe a n y induction of O D C activity or stimulation of [ H]-thymi3
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FIGURE 1. Schematic representation of vessel wall and some components thereof which may contribute to the pathogenesis of vascular disease. Matrix molecules: fibronectin (FN); laminin (LN); thrombospondin (TS); tenascin (TN); collagen (COL); heparan sulfate/heparin and elastin (HS). Growth Factors/Stimulators: epidermal growth factor (EGF); basic fibroblast growth factor (FGF ); platelet derived growth factor (PDGF); transforming growth factor β (TGF ); angiotensin (All) and low density lipopro tein (LDL). Furthermore, within the matrix compounds such as LDL and basic fibroblast growth factor can be bound. In the course of the normal turnover of matrix molecules which accompanies the dynamic process of tissue remodelling, peptide fragments having EGF domains can be generated and some of the stimulatory proper ties of this growth factor may become manifest. The compounds known to possess such EGF domains include LN, TS, and TN, a recently discovered matrix glycoprotein also found in vascular tissue. Both endothelial and smooth muscle cells are capable of secreting a number of growth factors without or with agonist stimulation, eg, Ang II. These events may lead to either hyper trophy or hyperplasia as well as the promotion of migration into the intimai space (see Discussion).
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Time after FCS addition (hrs) FIGURE 2. Kinetics of activation of important metabolic events leading to cell proliferation in VSMC from SHR and WKY rats. Quiescent cells were exposed to fetal-calf serum (10%) and the activity of S6 kinase, ODC, and the incorporation levels of [ H]thymidine into DNA determined (see Methods). The data repre sents the mean values from two experiments performed on a pair of SHR and WKY isolates at same passage number and at similar cell densities. Closed symbols represent data from SHR and open sym bols, WKY cultures.
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dine incorporation by this agonist w h e n a d d e d to serum-free m e d i u m . The VSMC from SHR a n d WKY sources elaborated extracellular matrices during long-term culture in pres ence of N a ascorbate, w h i c h consisted of a complex blend of connective m a c r o - m o l e c u l e s . Proliferation rates of WKY cells were differentially stimulated w h e n they were cultured u p o n matrices elaborated either by themselves or SHR cells in previous passage (Figure 3). G r o w t h rates of SHR cells were not significantly differ ent w h e n they were plated u p o n matrices elaborated by cells of either type (Figure 3). W h e n quiescent cultures of VSMC from SHR were exposed to A n g II there w a s a t i m e - d e p e n d e n t stimula tion of incorporation of [ C]-glycine a n d [ H]-glucosam i n e into extracellular matrix material above levels ob served for serum-free m e d i u m controls (Figure 4). Similar findings were obtained with cells from n o r m o tensive animals (data not shown). The data also sug gested a differential stimulation of glycoconjugate syn thesis by A n g II since the ratios for the incorporation of the t w o isotopic matrix precursors were significantly (P < .05) different, ( [ H ] / [ C ] ; 1.17 ± 0.2 for A n g II ν 0.77 ± 0 . 2 for controls). Although u n d e r serum-free conditions A n g II w a s incapable of stimulating mitogenic behavior in VSMC, in the presence of 1% P D S cells from SHR exhibited a n e n h a n c e d responsiveness to the agonist leading to a sig27
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FIGURE 3. Growth stimulation by extracellular matrix sub strata. VSMC were plated onto either gelatin-washed or matrixcoated substrata and grown for 3 days with normal medium. Following the growth period wells were trypsinized and cell num bers determined (see Methods). The data is expressed as the per centage of stimulation in growth (cell number) on the different matrix preparations (from SHR or WKY) relative to growth on gelatinized plastic (100°/o) and represents the means ± SD of re peated (n = 4) experiments. The hatched blocks represent data for SHR and * indicates the significance of difference (P > .001) in cell numbers for WKY when plated upon either SHR-derived ECM or their own. 18
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nificant (P < .001) sustained increase in cell n u m b e r (Figure 5). We also observed a minimal increase in prolif eration rates by cells from WKY rats exposed to A n g II a n d 1% P D S , w h i c h only reached significance (P < .01) after 9 days in culture. DISCUSSION VSMC from SHR sources exhibit elevated g r o w t h rates a n d e n h a n c e d responsiveness to g r o w t h stimulants (growth factors, h o r m o n e s , a n d FCS) as c o m p a r e d to cells from WKY s o u r c e s . ' W e h a v e also reported on the differential stimulation of S6 kinase in t h e t w o cell types as a consequence of their exposure to A n g I I . 1 8
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However, this agonist w a s u n a b l e to stimulate t h e acti vation of O D C or subsequent events leading to mitosis u n d e r serum-free conditions. We h a v e also s h o w n that other potential g r o w t h enhancers like low density lipo proteins (LDL) a n d t h r o m b o s p o n d i n (TS) b e h a v e in a similar m a n n e r . ' FCS, w h i c h contains a n u m b e r of mitogens for VSMC (ie, platelet derived g r o w t h factor (PDGF), basic fibroblast g r o w t h factor (FGF ), a n d epi dermal g r o w t h factor (EGF)) stimulated the induction of all three g r o w t h related metabolic events depicted in Figure 2. Cells from SHR r e s p o n d e d faster to FCS in terms of O D C activation a n d [ H]-thymidine incorpora tion t h a n did their normotensive (WKY) counterparts; this is a reflection of their faster g r o w t h rates in culture. Ang II stimulation of matrix glycoconjugate synthesis (Figure 4) m a y contribute to the structural changes that are part a n d parcel of vascular disease. The extracellular matrix plays an important role in the m a i n t e n a n c e of p h e n o t y p e , ' ' a n d our observations that matrix m a t e rial from SHR stimulated the g r o w t h of WKY cells to a greater extent t h a n that elaborated by themselves serves to emphasize the importance of this c o m p o n e n t of the vessel wall. We h a v e s u m m a r i z e d some of the current knowledge a n d some of our o w n findings in regard to the interactions of n o r m a l vessel wall c o m p o n e n t s (Fig ure 1). Central to these observations is the evidence that Ang II is capable of stimulating the expression of a n u m ber of important c o m p o u n d s w h i c h include P D G F A chain, TS, a n d T G F ^ . The induction of P D G F A chain expression in VSMC exposed to A n g II h a s already b e e n r e p o r t e d a n d it m a y account for our observation of g r o w t h stimulation by Ang II of cells from SHR in pres ence of 1% P D S (Figure 5). We h a v e found that cell 20
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isolates from SHR express a n d h a v e receptors for P D G F ^ h o m o d i m e r , w h e r e a s WKY-derived cells only express the g r o w t h factor b u t d o not respond to it, since they lack A-type cell surface r e c e p t o r s . The increase in cell n u m b e r observed in cultures from hypertensive an imals maintained in 1 % P D S m e d i u m alone m a y also be accounted for by their responsiveness to P D G F A-chain h o m o d i m e r . T h e synthesis a n d secretion of the AA homodimeric form of P D G F by arterial s m o o t h muscle cells h a s also b e e n observed by o t h e r s . The combina tion of vessel wall c o m p o n e n t s as depicted in the sche matic (Figure 1) can realistically lead to the initiation of neointimal formation w h i c h typifies vascular pathology in m a n a n d experimental animals. We h a v e found that c o m p o u n d s such as TS, LDL, a n d A n g II are able, in cultured VSMC, of e n h a n c i n g metabolic events nor mally associated with p r o l i f e r a t i o n . ' ' ' ' ' TS alone w a s not mitogenic to VSMC, but, w h e n adminis tered in combination with EGF, its mitogenic potential w a s considerably e n h a n c e d . ' A l t h o u g h there is n o evi dence as yet to suggest the presence of EGF (or its close analogue T G F ) in the normal vessel wall, recent data h a s s h o w n that laminin, w h i c h is certainly present, pos sesses EGF-like sequences within its s t r u c t u r e . It is conceivable that during the course of n o r m a l matrix turnover "EGF-like" molecules m a y arise a n d that these in combination with TS could b e h a v e as a strong mito genic mixture. Additionally, the stimulation of TGF^ ex pression by cultured s m o o t h muscle cells in response to A n g II e x p o s u r e m a y account for the influence of the vasoactive peptide on matrix synthesis, since TGF^ h a s b e e n s h o w n to not only m o d u l a t e the g r o w t h of smooth muscle cells b u t also to e n h a n c e their synthesis of extra34
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FIGURE 4. Kinetics of incorpora tion of radioactive matrix precursors into ECM macromolecules by VSMC from SHR. Quiescent cultures of SHR smooth muscle cells were ex posed to either [ C]-glycine or [ H]-glucosamine in the presence or absence of 100 nmol/L Ang II for the number of times shown. The determi nation of the levels of incorporation was performed as described under Methods and the data (mean values ± SD) is typical of that seen in a number of experiments (n = 4). Closed symbols represent cells maintained in the presence of Ang II and open, in its absence.
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Α.
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B.
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Majack RA, Goodman LV, Dixit VM: Cell surface throm bospondin is functionally essential for smooth muscle cell proliferation. J Cell Biol 1988;106:415-422.
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Scott-Burden T, Bühler FR: Regulation of smooth muscle proliferative phenotype by heparinoid-matrix interac tions. Trends Pharmacol Sei 1988;9:94-98.
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Naftilan AJ, Pratt RE, Dzau VJ: Induction of plateletderived growth factor Α-chain and c-myc gene expres sion by angiotensin II in cultured rat vascular smooth muscle cells. J Clin Invest 1989;83:1419-1424.
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Scott-Burden T, Resink TJ, Baur U, et al: Amiloride sensi tive activation of S6 Kinase by angiotensin II in cultured vascular smooth muscle cells. Biochem Biophys Res Commun 1988,151:583-589.
J
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FIGURE 5. Growth kinetics of VSMC from SHR and WKY in presence and absence of Ang II. Cells were plated into 12-well multiwell plates (1.25 X 10 cells/well) in normal medium con taining 10% whole blood serum. After 18 h medium was replaced with 1% PDS medium (see Methods) and some wells were further supplemented with Ang II (5X10 mol/L) on a daily basis. Me dium (1% PDS) was changed every 48 h and cell numbers deter mined as described previously. Experiments (n = 3) were per formed on cells from (A) SHR and (B) WKY and the data represent the means ± SD. Statistical significance was analyzed using Stu dent's t test for unpaired data and the levels of significance are indicated on the figure as *, Ρ < .01; **, Ρ < .001. 5
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cellular matrix m a c r o m o l e c u l e s . In conclusion, it w o u l d appear that w e n o longer h a v e to invoke d a m a g e to the e n d o t h e l i u m as the stimulus for the changes that lead to neointimal formation since m a n y c o m p o u n d s such as A n g II, alone or in combination with normal constituents of the vessel wall, are potentially capable of pathogenic modulation of medial s m o o t h muscle cells leading to progressive structural h y p e r t r o p h y , as o b served in vascular tissue from hypertensive animals a n d man.
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Berk BC, Vekstein V, Gordon HM, Tsuda T: Angiotensin II-stimulated protein synthesis in cultured vascular smooth muscle cells. Hypertension 1989;13:305-304.
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Campbell JH, Campbell GR: in Campbell JH, Campbell GR (eds): Vascular Smooth Muscle in Culture, Vol. 1. Boca Raton, Florida, CRC Press, 1987, pp 1 5 - 2 1 .
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