Induction of growth-related metabolism in human vascular smooth muscle cells by low density lipoprotein.

Human vascular smooth muscle cells (hVSMC) rendered quiescent by maintenance under serum-free culture conditions for 48 h exhibited several metabolic responses, normally associated with proliferation, following exposure to low density lipoprotein (LDL). LDL induced a time- and dose- (half-maximally effective concentration, ED50 25.0 +/- 8 nM) dependent activation of S6 kinase which could be negated following pretreatment of hVSMC with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h. In myo-[3H]inositol-prelabeled hVSMC, LDL caused a rapid (maximum within 1 min) decrease in phosphatidylinositol 4,5-bisphosphate (35% p less than 0.001) and phosphatidylinositol 4-phosphate (20%, p less than 0.01) with a return to prestimulated levels within 5-10 min. LDL induced a concomitant increase in [3H]inositol phosphates for which the order of generation was inositol-tris greater than -bis greater than -mono phosphate and which reached threshold levels of significance (p less than 0.05) above control values within 1, 2, and 10 min, respectively. The effect of LDL on hVSMC phosphoinositide metabolism was dose-dependent (half-maximally effective concentration, ED50 32.1 +/- 5.0 nM). This concentration, like that for S6 kinase, approximates with the KD (5-21 nM) for high affinity binding of 125I-LDL to specific receptors (1.5 x 10(4) sites/cell) on hVSMC. LDL induced a rapid but transient translocation of protein kinase C from the cytosol to membranes as assessed using both immunoblotting and [3H] 4-beta-phorbol-12-13-dibutyrate-binding procedures. Exposure of quiescent hVSMC to LDL elevated intracellular pH (delta pH 0.30 +/- 0.03, p less than 0.001). Such alkalinization was prevented in the presence of Na+/K+ exchange inhibitors such as amiloride, dimethylamiloride, and ethylisopropylamiloride. In an investigation of the nuclear action of LDL, a time-dependent induction of both c-myc and c-fos was observed. Such LDL-induced expression of these nuclear proto-onco-genes was not detectable in protein kinase C down-regulated hVSMC. Nevertheless, in spite of the cascade of "growth-promotional" responses elicited by LDL in quiescent hVSMC, this lipoprotein alone (under serum-free conditions) was neither mitogenic in nuclear labeling experiments, nor could it support growth of hVSMC in culture. We demonstrate that LDL might function in a complementary/synergistic fashion with other weakly mitogenic (to VSMC) growth factors and suggest that activation of protein kinase C (vis à vis intrinsic tyrosine kinase characteristic of other growth factor receptors) may be crucial to the signal transduction pathway for LDL.


Induction of Growth-related Metabolism in Human Vascular Smooth Muscle Cells by Low Density Lipoprotein*
Timothy Scott-Burden$, Therese J. Resink, Alfred W. A. Hahn, Ursula Baur, Rainer J. Box, and Fritz R. Buhler From the Department of Research,University Hospital,Basel, Human vascular smooth muscle cells (hVSMC) rendered quiescent by maintenance under serum-free culture conditions for 48 h exhibited several metabolic responses, normally associated with proliferation, following exposure to low density lipoprotein (LDL). LDL induced a time-and dose-(half-maximally effective concentration, EDso 25.0 f 8 nM) dependent activation of s6 kinase which could be negated following pretreatment of hVSMC with 12-O-tetradecanoylphorbol-13acetate (TPA) for 48 h. In myo-[3H]inositol-prelabeled hVSMC, LDL caused a rapid (maximum within 1 min) decrease in phosphatidylinositol 4,5-bisphosphate (35% p < 0.001) and phosphatidylinositol 4-phosphate (20%, p < 0.01) with a return to prestimulated levels within 5-10 min. LDL induced a concomitant increase in [3H]inositol phosphates for which the order of generation was inositol-tris >-bis >-mono phosphate and which reached threshold levels of significance ( p < 0.05) above control values within 1, 2, and 10 min, respectively. The effect of LDL on hVSMC phosphoinositide metabolism was dose-dependent (half-maximally effective concentration, EDso 32.1 f 5.0 nM). This concentration, like that for S6 kinase, approximates with the KO (5-21 nM) for high affinity binding of '2SI-LDL to specific receptors (1.5 X lo4 sites/cell) on hVSMC. LDL induced a rapid but transient translocation of protein kinase C from the cytosol to membranes as assessed using both immunoblotting and ['HI 4-&phorbol-12-13-dibutyrate-binding procedures. Exposure of quiescent hVSMC to LDL elevated intracellular pH (A pH 0.30 f 0.03, p < 0.001). Such alkalinization was prevented in the presence of Na+/K+ exchange inhibitors such as amiloride, dimethylamiloride, and ethylisopropylamiloride. In an investigation of the nuclear action of LDL, a time-dependent induction of both c-myc and c-fos was observed. Such LDL-induced expression of these nuclear proto-oncogenes was not detectable in protein kinase C downregulated hVSMC. Nevertheless, in spite of the cascade of "growth-promotional" responses elicited by LDL in quiescent hVSMC, this lipoprotein alone (under serumfree conditions) was neither mitogenic in nuclear labeling experiments, nor could it support growth of hVSMC in culture. We demonstrate that LDL might function in a complementary/synergistic fashion with other weakly mitogenic (to VSMC) growth factors and suggest that activation of protein kinase C (vis ti vis intrinsic tyrosine kinase characteristic of other growth * This work was supported by Swiss National Foundation Grant 3.924.083. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed.
factor receptors) may be crucial to the signal transduction pathway for LDL.
The stimulation of quiescent cells in culture to proliferate by serum and peptide growth factors has been shown to be dependent on a number of coordinated cellular events leading to DNA synthesis and cell division (1)(2)(3)(4). One such event is the stimulation of s6 kinase activity which results in the reactivation of protein synthesis and is a prerequisite for cell division (5-7). We have previously studied s 6 kinase activation (8)(9)(10) in vascular smooth muscle cells (VSMC)' and have shown that some compounds, i.e. thrombospondin and angiotensin I1 (AII), which are not normally considered as growth factors for such cells, are capable of stimulating protein synthesis (8,9). We have also confirmed the findings of Pouyss6gur et al. (ll), namely that s6 kinase activation and elevation of intracellular pH are intimately associated (9). However, although in our hands neither AI1 nor thrombo spondin were mitogenic to VSMC under serum-free conditions, both compounds were capable of eliciting a number of important proliferative intracellular responses suggesting that in combination with other factors they may be growth promoters in uiuo. In particular, AI1 has been shown to stimulate expression of the c-fos proto-oncogenes in VSMC in an analogous manner to fetal calf serum (12), and we and others have confirmed that thrombospondin in combination with EGF markedly stimulates DNA synthesis in VSMC (13,14). There has been a prolonged interest in the role of low density lipoproteins (LDL) in VSMC proliferative behavior (15)(16)(17)(18). Some evidence suggests that LDL is mitogenic to VSMC (16) while other studies have shown that the same compound only supplies essential lipids for growth (18). These discrepancies mostly relate to the culture conditions/treatments under which the effect of LDL was tested. More recently, Chen et al. (19) observed stimulation of growth of VSMC only by intact LDL particles and not the separate lipid and/or apoprotein components, which suggests to US that LDL may indeed play an important promotional role to VSMC proliferation.

Methods
Isolation and Culture of Vascular Smooth Muscle Cells-The isolation and culture of human vascular smooth muscle cells (hVSMC) was performed essentially as described previously for rat VSMC (20); the tissue of origin was obtained from patients undergoing abdominal surgery and consisted of microarterioles associated with omental fat. Tissue was extensively cleaned to remove all connective tissue by micro dissection and then finely minced prior to enzymatic dissociation (20). Cell isolates were plated onto extracellular matrix-coated plastic ware (21, 22) and cultured as described (20). When primary cultures reached confluence (7-9 days, depending on seeding density) cells were redispersed by trypsinization and fresh cultures initiated on gelatine-coated plastic ware. Cells were phenotypically characterized in second passage as described previously (21) and checked for absence of factor VI11 antibody utilizing bovine endothelial cells as positive controls. Experiments described here used two isolates of (H6 and HID) hVSMC between passage 4 and 12 between which the parameters investigated remained steady. Prior to all experimentation confluent hVSMC were rendered quiescent by culture for 48 h (with one medium change after 24 h) in serum-free medium containing 0.1% (w/v) bovine serum albumin instead of 20% fetal calf serum.
Preparation and Iodination of Human Low Density Lipoprotein-LDL (density 1.019-1.063) was prepared as described by others (18, 23) from human plasma using sequential ultracentrifugation and bouyant density centrifugation techniques. Potassium bromide solutions were used for density adjustments (24), and isolated LDL fractions were exhaustively dialyzed against phosphate-buffered saline in the presence of anti-oxidants (18) prior to sterilization by filtration through 0.45 PM Gelman filters. Samples were stored a t 4 "C and protein concentration determined. LDL was iodinated (specific activity 5-6 pCi/nmol) by the iodine monochloride method as described by Bilheimer et al. (25). Saturation binding of '251-LDL (0.5-150 pg/ml) to hVSMC was performed at 4 "C for 3 h in 0.5 ml of Hepes-buffered Dulbecco's modified Eagle's medium essentially as described by Aulinskas et al. (23). A parallel series of dishes also contained excess (2.25 mg/ml) unlabeled LDL for determination of nonspecific binding. Cell-bound IZ5I-LDL was determined following solubilization in 0.5 ml of 1% sodium dodecyl sulfate and counting in a y-counter.
Some dishes were preincubated with I,GC7 (10-50 nM) for 2 h at 4 "C prior to saturation binding assays (26). Control dishes were also maintained at 4 "C for 2 h in the presence of monoclonal antibody to factor VI11 antigen, a procedure which did not reduce specific binding Assay of s 6 Kinase Activation in hVSMC-The assay of S g kinase activation was performed exactly as described before (a), following exposure of quiescent hVSMC to agonists for times and doses described under the relevant "Results" section. The level of stimulation of S g kinase by 10% FCS was always determined as a positive control for each experiment.
Assays of Phosphatidylinositol Phosphate Catabolism-The LDLinduced catabolism of hVSMC phosphatidylinositol phosphates was determined in cultures prelabeled for 48 h and with my~-[~H]inositol (5 pCi/ml culture medium) under serum-and inositol-free conditions. Experimental procedures for stimulation (in the presence of 15 mM LiCl) of phosphoinositide turnover and for extraction and resolution of metabolites thereof were as described previously for rat VSMC (27).
Translocation of Protein Kinase C and [JHIPDBu Binding-After exposure of quiescent hVSMC to LDL (50 pg/ml) for required time intervals dishes (100-mm Petris, lo7 cells/dish) were washed rapidly three times with phosphate-buffered saline at 4 "C and maintained on ice. 700 pl of extraction buffer (PKB: containing 20 mM Tris-HC1, pH 7.4,2 mM EGTA, 10 mM 0-mercaptoethanol, 20 pg/ml leupeptin, 2 pg/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) was added to each dish, cells were harvested, and disrupted by sonication. 200-p1 aliquots were withdrawn (homogenate) and the remainder centrifuged at 100,000 X g for 30 min at 4 "C. Supernatants (cytosol) were withdrawn before suspension and sonication of pellets (membrane) in 500 p1 of PKB. Samples were stored at -70 "C until use. Phorbol ester binding was performed essentially as described previously (31) in the presence and absence of 4 FM PDBu. Sample aliquots were incubated (overnight at 4 "C with vigorous shaking) in a reaction mixture (250 p1) containing 20 mM Tris-HC1, pH 7.4, 10 mM Mg(NO&, 1 mM CaCL, 400 pg/ml phosphatidylserine, 4 mg/ml bovine serum albumin, and 50 nM [3H]PDBu, and thereafter [3H] PDBu binding determined by filtration through Whatman GF/C filters and washing with ice-cold buffer containing 20 mM Tris-HC1, pH 7.4, 10 mM Mg (NO,),, 1 mM CaC12 (31, 32). Specific binding was estimated as total bound minus that bound in the presence of 4 PM PDBu. Data are expressed as the percentage distribution of [3H] PDBu between cytosol and membrane fractions where that bound in the homogenate fraction was taken to represent total (100%). The appropriate corrections for assay and extraction volumes were made.
In a separate series of experiments, hVSMC were treated with LDL (50 pg/ml), or TPA (1 nM), or 4 a-phorbol-12,13-didecanoate (1 of lZ5I-LDL. nM) for the required intervals. Cells were harvested, sonicated, and centrifuged as described above. The membrane fractions were dissolved at 95 "C in 1% SDS, 10 mM Tris-HC1, pH 6.8, 0.05% 0mercaptoethanol, 50% glycerol, subjected to sodium dodecyl sulfatepolyacrylamide (10%) gel electrophoresis and transferred electrophoretically to nitrocellulose blotting membranes (Bio-Rad) (33). Membranes were processed for immunoblotting exactly as described (31) using monoclonal antibody to protein kinase C (clone MC5, Amersham Corp.) and 0.2 pCi/ml '252-labeled (Amersham Corp. 18 pCi/pg) sheep-anti-mouse IgG as the second antibody. Immunodetectable protein kinase C (80 kDa) was quantitated after location by autoradiography, excision of material from membranes, and counting in a multiwell y-counter. All values were normalized with respect to protein (30-40 pg) loaded on the gels. Measurement of Proto-oncogene Induction-Quiescent hVSMC, grown in 150 mM dishes, were exposed to either FCS (20%), LDL (100 nM), or TPA (100 nM) for times indicated in the relevant results section. Stimulated cells were harvested by trypsinization. After a single wash in phosphate-buffered saline cells were lysed in GTbuffer (34) and RNA was collected by centrifugation of the lysate on a 5.7 M CsCl gradient at 35,000 rpm in an SW50 rotor for 12-16 h (34).
For Northern blotting 20 pg of total RNA was electrophoresed through an 1.2% Agarose gel containing 2.2 M formaldehyde. Gels were run at 50 V for 6-8 h in MOPS buffer and blotted to Hybond nylon membranes (Amersham Corp.) with 20 X SSC (sodium chloride, sodium citrate) as blotting buffer. Subsequently, membranes were removed, washed briefly in 2 X SSC, dried for 10 min at 80 "C, and then RNA was fixed to membranes by UV irradiation for 3 min at 302 nm. Blots were hybridized to random primed c-myc (35) and cfos (36) probes according to the method of Gilbert and Church (37). Exposure of blots to Kodak X-Omat films was done overnight at -70 "C with the use of one intensifying screen.
Assay for DNA Synthesis-Assays of DNA synthesis were performed on quiescent hVSMC (serum deprivation for 48 h) plated into costar 12-well multiwell dishes as described previously (38) using the method of Bradford (29), with bovine serum albumin as standard.

RESULTS
Specific Binding of LDL to Intact Cells-Quiescent human vascular smooth muscle cells (hVSMC) exhibited specific and saturable binding for lZ5I-labeled LDL (Fig. 1). Scatchard transformation of the data (inset, Fig. 1) indicated that hVSMC possessed high affinity (KO ranged from 0.5 to 2.1 X M in three separate experiments) and low affinity (KO > M ) sites. The number of high affinity LDL receptors found on hVSMC in three separate experiments was -1.5 X IO4 sites/cell.
Preincubation of cultures with C7 monoclonal antibodies (I,GC7) against specific LDL surface receptor (26) reduced subsequent binding of 1251-labeled LDL by -60%. I,GC7 was used at between 10 and 50 nM in three binding experimental protocols.
Activation of Ss Kinase by LDL-LDL induced a timedependent activation of Ss kinase in quiescent hVSMC (Fig.  2). The time course for this activation was similar to that previously observed following exposure of quiescent rat aortic VSMC (9), or hVSMC to TPA (not shown). These kinetics are characterized by a relatively slow maximum after (30 min) and more sustained activation of Ss kinase as opposed to the rapid maximum (after 10 min) and transient activation profiles observed in response to growth factors (e.g. EGF (7,8 ) ) and vasoconstrictors (e.g. AI1 (9)). LDL-induced activation of Ss kinase was dose-dependent and the half-maximally effective concentration of LDL was calculated to be 25.0 f 8 nM (Fig. 3A). This is comparable to the KD value obtained for LDL binding to high affinity sites. The ability of TPA to activate Ss kinase in quiescent hVSMC was negated when   cells were first pretreated for 48 h with this phorbol ester ( Fig. 3B). Such a pretreatment (prior to exposure to LDL) also abolished any stimulatory effects of LDL on Ss kinase (Fig. 3A). The data in Figs. 2 and 3 thus strongly suggested that LDL-induced stimulation of s 6 kinase might be mediated via protein kinase C since this enzyme specifically is considered to be the cellular phorboid receptor (39, 40).
Since protein kinase C is dependent on diacylglycerol (and Ca2+) for activation, its participation in the signal transduction pathway for LDL would imply hydrolysis of phosphatidylinositol-bisphosphate and consequent generation of inositol trisphosphate and diacylglycerol (40, 41). We therefore investigated the ability of LDL to elicit catabolism of phos-phoinositides in my~-[~H]inositol prelabeled VSMC. Fig. 4 shows the changes in three [3H]inositol phospholipids (Ptd-Ins, Ptd-InsP, Ptd-InsP2) of hVSMC at various times after addition of LDL. There was a rapid, transient decrease in 3H content of Ptd-InsP (by -20%, p < 0.01 after 1 min) and Ptd-InsPz (by -35%, p < 0.002 after 1 min) with a return to prestimulated levels within 5-10 min. There was little change in Ptd-Ins with 3H content decreasing to significance (-lo%, p < 0.05) only after 15 min of exposure to LDL. observed a rapid but transient increase of immunoreactive protein (Mr 80,000) in the membranes isolated from hVSMC following exposure to LDL (Fig. 6). With prolonged exposure to LDL, immunoreactivity to anti-protein kinase C progressively decreased to levels found in membranes from control, quiescent hVSMC. The extent of translocation induced by TPA was 3-fold greater than for LDL, and was protracted (Fig. 6).
On the assumption that protein kinase C is the specific receptor for phorbol ester (31, 39, 41) the following experiments confirm that LDL-induced translocation of protein kinase C from the cytosol to the membrane in hVSMC. At quiescence the distribution (as % of total in homogenate) of [3H]PDBu binding between membrane and cytosol fractions was 38 f 5% and 70 k 6%, respectively (Fig. 7). Following exposure to LDL the % distribution of [3H]PDBu in membranes increased with a concomitant and inversely proportional (% decrease) change in cytosolic 13H]PDBu binding (Fig. 7). Such translocation induced by LDL was maximal within 2 min whereafter a retranslocation of [3H]PDBu binding occurred which remained stable with prolonged exposure (up to 30 min) to LDL.
Amiloride-sensitive Alkalinization-Both protein kinase C and s6 kinase activation are intimately involved in the onset of proliferation of quiescent cells, and their stimulation is associated with a rapid intracellular alkalinization brought about via activation of the Na+/H+ antiporter (4,11,39,42,44). Exposure of quiescent hVSMC to LDL elevated intracellular pH with the ApH, being similar to that measured in the presence of 10% FCS (Table I). TPA but not the 4,a-phorbol isomer also increased pHi. The effects of LDL on hVSMC pH, were prevented in the presence of Na+/H' exchange inhibitors such as amiloride and amiloride analogues, DMA or EIPA (Table I) stimulation procedures at 37 "C (see Methods). Smooth muscle cells have been shown to rapidly internalize bound C7 antibodies at 37 "C (26). The level of inhibition by C7 of the signal transduction processes studied was comparable to that observed for '251-labeled LDL binding to antibody-treated cells (results above -LDL binding). When LDL (10-50 pg of protein) was preincubated (4 "C for 2 h) with antiapo B (12 pg of protein) prior to addition to quiescent hVSMC in stimulation assays (phosphoinositide turnover and Sg kinase activation) we observed a decreased (range 60-28% of control values) level of induction of these pathways as compared with control cultures treated with antiapo B only.
The levels of stimulation of phosphoinositide metabolism and Ss kinase activation by AI1 in cultures of quiescent hVSMC were not reduced by either antibody preparation.
Nuclear Proto-oncogene Expression, Mitogenesis, and Growth-The data thus far presented demonstrate that in hVSMC, LDL elicits several biochemical responses common to the action of a variety of recognized growth factors (41, 44). Increasing attention is being focused on the possible role for hormones in regulating cell proliferation, and of particular  interest to VSMC in this regard is the vasoconstrictor angiotensin 11. In addition to the ability of AI1 to simulate phosphoinositide catabolism and elevate intracellular pH (27,45), this hormone has recently been demonstrated to activate Se kinase (9) and to induce expression of the nuclear protooncogenes c-myc and c-fos (12,46). We have thus investigated the nuclear actions of LDL. Exposure of hVSMC to LDL resulted in a time-dependent stimulation of both c-myc and c-fos expression, although the levels of expression were somewhat smaller than those induced by 10% (v/v) FCS (Fig. 8).
The time courses for induction were identical for TPA and LDL. After pretreatment of hVSMC with TPA for 48 h, a time-dependent induction of c-myc expression by LDL was not discernable. However, some induction of c-fos remained under these conditions. The induction of the two protooncogenes by re-exposure of pretreated cells to TPA was also not apparent (Fig. 8B). Pretreatment of quiescent cultures with TPA did not, however, adversely affect the inductive capacity of 10% (v/v) FCS (Fig. 8B).
To investigate the growth promotional properties of LDL, hVSMC were subjected to long-term exposure to LDL under serum-free conditions. No increase in cell number was evident and the stimulation of [3H]thymidine incorporation into DNA was very small by LDL alone (Fig. 9). However, in combina-tion with EGF, itself a weak mitogen for VSMC (13), we observed both sustained, albeit slow, growth of hVSMC and a significant stimulation of DNA synthesis (Fig. 9). Indeed combinations of LDL and EGF resulted in a synergystic stimulation of DNA synthesis (compare LDL/EGF, with A ; Fig. 96).

DISCUSSION
This study demonstrates that LDL is capable of activating, in human vascular smooth muscle cells, a number of cellular events which are intimately linked to the onset of proliferation in quiescent cells (41,44). These processes include phosphoinositide turnover, activation of s6 kinase, alkalinization, translocation of protein kinase C, and induction of nuclear proto-oncogene expression.
Furthermore using specific antibodies to either LDL (antiapo B) or its cell surface receptor, I,GC7 (26) we have been able to demonstrate that the observed responses are specific to LDL and are receptor mediated. To obtain complete inhibition of the LDL stimulations of hVSMC using the C7 receptor antibody was difficult since the latter is rapidly internalized at 37 "C during incubation of cells with LDL for stimulatory purposes. Nevertheless, the 60% reduction in binding of lZ5I-labeled LDL to I,GC7-treated cells was paralleled by a similar reduction in their stimulation (S6 kinase, phosphoinositide turnover) by LDL. Prior treatment of LDL preparations with monoclonal antibody to platelet-derived growth factor at levels that negated responses due to this growth factor (8) did not reduce the stimulatory effects of LDL on hVSMC in our studies. We have also shown that platelet-activating factor used at levels that have been shown to be present in LDL preparations of others (47) has no stimulatory effects on the second message transduction processes studied by us (38).
Our findings with respect to the catabolic effects of LDL on phosphoinositide metabolism confirm those made in a previous study on human platelets, lymphocytes, lung fibroblasts, and rat aortic VSMC, and in which LDL was additionally shown to elicit increases in both intracellular free Ca*+ concentrations and diacylglycerol production (48). Presumably the LDL-induced increase in Ins-P3 also promotes Ca2+ release from intracellular stores (40) in human VSMC. Production of diacylglycerol is implicit from the catabolism of polyphosphoinositides (40,41), and the transient nature of this metabolite is supported by the transient nature of protein kinase C membrane association observed with both immunoblotting and [3H]PDBu-binding experiments. TPA however, which is not readily degraded enzymatically (vis a vis rapid conversion of diacylglycerol to phosphatidic acid and arachidonic acid (41)) induced a protracted association of protein kinase C with membranes (this study and 42, 49, 50). Cytosolic protein kinase C is normally functionally inactive whereas following agonist stimulation and generation of diacylglycerol the enzyme translocates to the membrane where the lipid-rich environment plus Ca2+ serve as cofactors for enzymatic kinase activity (40,42,49,50). The ability of LDL to cause phosphoinositide degradation and a concomitant transposition of protein kinase C, therefore, suggests that activation of this enzyme may be crucial to the signal transduction pathway for this lipoprotein.
The activation of S, kinase in response to agonists is recognized to be dependent on multiple phosphorylation of this enzyme itself. This can be mediated by both a tyrosine kinase-initiatedpathway (an activity intrinsic to many growth factor receptors (41)) and/or by protein kinase C (3,4,7,9). Evidence for the specific involvement of protein kinase in the activation of s 6 kinase in hVSMC by LDL was obtained from experiments in which cells were exposed to TPA for 48 h prior to lipoprotein administration. Such prolonged exposure results in down-regulation of protein kinase C and a concomitant loss of phorbol ester responsiveness (this study and 49, 51). We found that down-regulation of protein kinase C in hVSMC completely negated subsequent induction of S 6 k' lnase activation not only by TPA but also by LDL. Furthermore, induction of c-fos and c-myc expression by LDL was also found to be largely absent in hVSMC pre-exposed to TPA (Fig. 8). Such data contrast with observations made for other growth factors (8) and hormones (9) which also elicit phosphoinositide catabolism but whose stimulatory effects, at least on s 6 kinase, are not decreased in TPA down-regulated cells.
The ability of TPA to stimulate the Na+/H+ antiporter (this study and 52) and the elevation of intracellular pH in response to numerous growth factors/hormones which elicit phosphoinositide degradation (41, 43-45, 53) have invoked the involvement of protein kinase C in cytoplasmic pH regulation. Since intracellular alkalinization is a prerequisite for agonist-induced activation of s6 kinase (4, 9, ll), it is not surprising that LDL also elevated intracellular pH in hVSMC. Our experiments with amiloride and the amiloride analogues, DMA and EIPA, indicated that the increase in pH is mediated via stimulation of the amiloride-sensitive Na+/H+ antiporter as has been found for TPA and growth factors/hormones (4,9,11,41,43,45,52). Such a metabolic response is believed to play a significant role in induction of cell proliferation and division (39, 41), phosphoinositide signal transduction (53), and regulation of muscle contractility and tone (43).
Our results further substantiate the findings of others in relation to nuclear proto-oncogene activation in cultured VSMC by mitogenic agents (36, 54) and also underlines the potential growth promotional properties of LDL. The observation that the time course for c-myc induction by LDL mimicked that of TPA (Fig. 8) and differed substantially from that of FCS and other mitogenic compounds make it unlikely that the effects of LDL reported here are due to contamination of our preparations with such agents. Although we observed a low constitutive expression of c-myc in our cultures maintained on serum-free medium, we are unable to say if such transcripts are functionally active. They may represent the pool of more stable, non-polyadenylated c-myc mRNA molecules recently reported by others (55). The detailed mechanism of c-fos induction is still not fully elucidated, although in the case of FCS it appears that specific serum response elements in the promoter region of the gene are important for this process (56,57). The same is not true for c-fos activation by TPA, working as it does through protein kinase C (58). The fact that we still observed some residual activation of cfos by LDL following prolonged pretreatment of hVSMC with TPA suggests that either an alternative pathway exists for LDL to exert this effect, or activation of c-fos occurs as a consequence of stimulation of other intracellular events by LDL as already discussed above.
In spite of the ability of LDL to promote such a wide spectrum of characteristic mitogenic cellular responses, we found that LDL alone is neither mitogenic to hVSMC nor would it support these cells in culture over a long period (5-7 days) under serum-free conditions. Clearly, therefore, it cannot be defined as a true mitogen for VSMC, but that this lipoprotein does elicit a cascade of growth promotional responses would suggest that its role is more important than mere supply of lipids essential for growth (18).
We propose and our data (Fig. 9) support the idea that LDL may play a complimentary role with other growth factors along the lines already shown for thrombospondin with epidermal growth factor (13, 14). Thrombospondin alone, like LDL, will not promote growth or appreciably stimulate DNA synthesis but in the combined presence of epidermal growth factor, a weak mitogen for VSMC, a marked synergistic proliferative response is observed (13,14). Our findings with combinations of LDL and EGF ( Fig. 9 and Ref. 38) lend further credence to the importance of growth factor/growth promoter complementation. Such interactions have also been proposed for oncogenes (41). We are currently investigating possible mitogenic interactions between LDL and other wellknown mild VSMC mitogens.
A fundamental question remaining concerns the importance of the stimulatory properties of LDL reported herein in relation to the inappropriate and deregulated growth of VSMC in vascular pathology. Evidence is accumulating for the trapping and localized concentration of LDL within the extracellular milieu of the blood vessel wall (59-61). The relevance of the ability of LDL to activate proliferative responses may reside in this area because of the influence that matrix macromolecules exert on several cellular processes including proliferation (62). Furthermore, the once simplistic view that growth factors were specific for a single cell type and could per se affect a proliferative influence on that target cell has long been replaced by the concept of a complex orchestrated interplay between growth promoters and inhibitors, many of whose actions may depend on interactions at sites removed from their specific cell surface receptors (44, 63, 64).