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J Am Coll Cardiol, 1999; 34:1644-1651
© 1999 by the American College of Cardiology Foundation
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EXPERIMENTAL STUDIES

Vascular smooth muscle proliferation

Synergistic interaction between serotonin and low density lipoproteins

Shinji Koba, MD*, Rajbabu Pakala, PhDa, Takuya Watanabe, MD*, Takashi Katagiri, MD* and Claude R. Benedict, MD, DPhil, FACCa

a Department of Internal Medicine, Division of Cardiology, University of Texas Health Science Center at Houston, Houston, Texas, USA
* Showa University School of Medicine, Tokyo, Japan

Manuscript received September 21, 1998; revised manuscript received May 18, 1999, accepted June 28, 1999.

Reprint requests and correspondence: Dr. Claude R. Benedict, Department of Internal Medicine, Division of Cardiology, The University of Texas Health Science Center, 6431 Fannin, MSB 6.039, Houston, Texas 77030


   Abstract
Top
 Abstract
Materials and methods
 Results
 Discussion
 References
 
OBJECTIVES

The purpose of this study was to examine whether low density lipoproteins (LDLs) or mildly oxidized LDL (mox-LDL) are mitogens for vascular smooth muscle cells (VSMCs) and whether they can act synergistically with serotonin (5HT), a known mitogen for VSMC, in potentiating the proliferative effect of 5HT on VSMC.

BACKGROUND

Whether LDL or mox-LDL has a mitogenic effect on VSMC has been controversial. It is possible that LDL may not be mitogenic to VSMC but modification of LDL may confer mitogenic properties on LDL. A known mitogen for VSMC is 5HT that is released by aggregating platelets at sites of atherosclerotic changes or endothelial dysfunction. It is possible that LDL may interact with 5HT to enhance VSMC proliferation induced by 5HT.

METHODS

Growth arrested primary VSMCs were incubated with different concentrations of LDL or mox-LDL for 24 h followed by incubation with 5HT for another 24 h (mild oxidation of LDL was achieved by incubating LDL with Cu++ which increased the thiobarbituric acid product formation without a change in electrophoretic mobility). The increase in cell number or the amount of 3H-thymidine incorporated into the DNA was then measured.

RESULTS

Low density lipoprotein and mox-LDL induced significant VSMC proliferation by themselves and this effect was potentiated by 5HT. The 5HT2 receptor antagonist (LY281067) and pertussis toxin reversed only the proliferative effect of 5HT. Polyinosinic acid (poly-I), an inhibitor of scavenger receptors, did not inhibit the proliferative effect of LDL or mox-LDL or their synergistic interaction with 5HT.

CONCLUSIONS

These results suggest that LDL and mox-LDL act synergistically with 5HT in inducing VSMC proliferation. The synergistic interaction could be blocked by LY281067 and pertussis toxin but not by poly-I acid.

Abbreviations and Acronyms
  BSA = bovine serum albumin
  DMEM = Dulbecco’s modified Eagle’s medium
  EDTA = ethylenediamine tetraacetic acid
  FBS = fetal bovine serum
  HBSS = Hank’s balanced salt solution
  JNK/SAPK = c-Jun N-terminal kinases/stress activated kinase
  LDL = low density lipoprotein
  Lyso-PC = lysophosphatydilcholine
  MAP-kinase = mitogen activated protein kinase
  mox-LDL = mildly oxidized low density lipoprotein
  PBS = phosphate buffered saline
  poly-I = polyinosinic acid
  PTX = pertussis toxin
  SMC = smooth muscle cell
  TBARS = thiobarbituric acid reactive products
  VSMC = vascular smooth muscle cell
  5HT = serotonin

Proliferation of intimal vascular smooth muscle cells (VSMCs) is an important component in the development of atherosclerosis and restenosis after angioplasty (1–3). Low density lipoproteins (LDL) are the major cholesterol-carrying lipoproteins, and the association between high levels of plasma LDL and the development of atherosclerosis is well-established. However, whether LDL directly promotes the proliferation of VSMCs is controversial (4–15). It is known that oxidative modification of LDL plays an important role in the atherogenic process. Oxidation of LDL is progressive, leading initially to the formation of mildly oxidized LDL (mox-LDL) which is defined by a low content of lipid peroxidation derivatives and little modification in apolipoprotein B. Further modification leads to the formation of oxidized LDL which contains high levels of lipid peroxidation products and significant alterations in apolipoprotein B (16–20). Highly oxidized LDL is known to be cytotoxic for a variety of cells including VSMC, whereas mox-LDL is a mitogen for vascular endothelial cells and VSMCs (12–13,21). This suggests that the biological properties of mox-LDL may be different from those of oxidized LDL (20).

The presence of both activated platelet aggregates and lipoproteins in the atherosclerotic plaque suggests that both may participate in the pathogenesis of the atherosclerotic lesion formation (22). Activated platelets release various mediators, including peptide growth factors such as beta-thromboglubin and Platelet Derived Growth Factor from alpha granules and nonpeptide products such as serotonin (5HT), adenosine diphosphate and adenosine triphosphate (22) from dense granules. Serotonin mediates vasoconstriction and induces activation of other platelets, which may promote the progression of coronary arterial diseases by adhering to unstable atherosclerotic plaque(s) and initiating thrombotic complications (23–25). Studies have shown that 5HT is a mitogen for VSMCs (26–28), which may contribute to the development of atherosclerotic lesions or neointimal proliferation after vascular injury such as angioplasty. When atherosclerotic plaque ruptures, either as part of natural progression of disease or after angioplasty, VSMCs are exposed to flowing blood in circulation. Because platelets aggregate and release 5HT at the site of injury, the interaction of 5HT with lipoprotein particles from plasma may act in concert to stimulate the proliferation of arterial SMCs (29). In addition, both native and oxidized LDL increase the releases of 5HT from platelets and amplify 5HT-induced platelet aggregation at physiologic concentrations (22,23,25,30–32). Recent reports have also shown that mox-LDL can induce platelet aggregation and secretion (33). However, whether there is an interaction between 5HT and LDL or mox-LDL on inducing VSMC proliferation is unknown. In this study we report that LDL and mox-LDL are mitogenic to VSMCs and they act synergistically with 5HT in inducing VSMC proliferation.


   Materials and methods
Top
 Abstract
 Materials and methods
Results
 Discussion
 References
 
Materials.   Serotonin (as creatine sulfate), bovine serum albumin (BSA), insulin, transferrin, pargyline and pertussis toxins were purchased from Sigma (St. Louis, Missouri). The LY281067 was a gift from Eli Lilly Laboratories. Serotonin was dissolved in 50 mM citrate buffer pH 5.5. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and phosphate buffered saline (PBS) was purchased from Gibco BRL Life Technologies (Gaithersburg, Maryland). Trypsin ethylenediamine tetraacetic acid (EDTA) and Hank’s balanced salt solution (HBSS) were purchased from Sigma and 3H-thymidine (specific activity 20Ci/mol) was from Dupont NEN Research Products, Boston, Massachusetts. Other reagents were purchased from local vendors.

Arterial smooth muscle cell isolation.   Rabbit primary aortic VSMCs were isolated using the explant method (28). The intima was first peeled off from the aorta and then the media carefully stripped away from the adventia and placed in a petri dish containing warmed HEPES-buffered DMEM (37°C). The medial layer was cut into approximately 1 mm2 squares, which were transferred to a 25 cm2 tissue culture flask and barely covered with DMEM supplemented with 20% FBS. The blocks of tissue were cultured at 37°C in a humid atmosphere of 5% CO2 and 95% air (vol/vol). After three to four weeks the tissue blocks were removed and the migrated VSMCs were cultured, followed by subculture using trypsinization. The identity of the VSMCs was confirmed by morphologic examination and by staining for alpha-actin.

Isolation and preparation of LDL.   Low density lipoprotein (density 1.019–1.063) was obtained from plasma containing 1mM ethylenediamine tetraacetic acid (EDTA) by density-gradient ultracentrifugation using the method of Ferrei (34). Solid Kbr was used to adjust the density, and centrifugation was carried out at 55,000 rpm for 15 h at 5°C (Beckman 70.1 Ti rotor, Palo Alto, California). To minimize variation in experimental results due to differences in plasma donors, plasmas were pooled from several animals. Isolated fractions were dialyzed at 4°C against PBS using 6,000 to 8,000 molecular weight cut off dialysis membrane for 48 h and then concentrated under nitrogen using Amicon concentrator with 10 KD cut off membrane.

Low density lipoprotein (1 to 2 mg/mL) was mildly oxidized at 37°C in the presence of CuSO4 at a final concentration of 5 µmol/L for 24 h. The oxidation was terminated by the addition of EDTA (1.0 mmol/L, final concentration). Minimally oxidized LDL fractions were dialyzed against PBS with EDTA (0.1 mmol/L, final concentration) and then concentrated in the same manner.

Experiments were carried out using physiologically relevant (concentrations tested are found in circulation in patients) and most commonly (experimentally) described (20–200 µg/ml) concentrations of lipoproteins. Lipoprotein concentrations are expressed as protein concentrations, which were determined by the Bradford method using BSA as standard (35). The purity and the extent of the oxidation of LDL were determined by 1% agarose gel electrophoresis and thiobarbituric acid reactive products (TBARS) formation. Thiobarbituric acid reactive products were measured by mixing 100 µg of LDL or mox-LDL with 3 mL of mixed solution consisting of 15% tricholoracetic acid, 0.375% thiobarbituric acid and 0.25 N hydrochloric acid. This mixture was boiled for 15 min, centrifuged and the absorbance of the supernatant measured at 532 nm using tetramethoxy propane as a standard (36). Even at a concentration of 10 mg/ml, native LDL had little or no effect on TBARS. The mox-LDL had 29.9 ± 2.6 pmol/mg protein of TBARS formed, but it exhibited little or no change in the electrophoretic mobility. The prepared LDL was used within 30 days of isolation. Lipoproteins were sterilized by a 0.22 micron filter before use.

3H-thymidine incorporation.   Primary VSMCs from first or second passage were seeded into 35 mm diameter tissue culture plates and grown to semiconfluence in DMEM containing 10% FBS. Then, the growth medium was replaced with 2 ml DMEM containing 0.1% FBS and was incubated for approximately 72 h for growth arresting and synchronization. The medium was then replaced with DMEM supplemented with 500 µg/ml BSA, 10 µg/ml bovine insulin, 20 µg/ml human transferrin, 25 ng/ml selenium, 100 µmol/l parglyine and the indicted concentrations of native LDL or mox-LDL. After 24 h of incubation, the given concentrations of 5HT were added and incubated for another 24 h. Tritiated thymidine at a concentration of 1 µCi/plate was added to the medium for the last 5 h. At the end of this period the medium was removed, and the plates were washed with ice-cold PBS. At that point, 6% trichloroacetic acid was added to the cells, and the acid-insoluble 3H-thymidine was collected on glass fiber filters. The filters were washed with 100% ethanol and air dried, and 3H-thymidine was quantified with a liquid scintillation counter. All experiments were performed in quadruplicate and each experiment was repeated a minimum of three times.

Determination of VSMC number.   Primary VSMCs from first or second passage were seeded into a 35 mm diameter plate at a density of 85,000 to 120,000 cells per plate in DMEM containing 10% FBS. After 96 h, growth medium was replaced with 2 ml DMEM containing 0.1% FBS and incubated for approximately 72 h for arresting cell growth and synchronization. Then, the medium was replaced with DMEM supplemented with 500 µg/ml BSA, 10 µg/ml bovine insulin, 20 µg/ml human transferrin, 25 ng/ml selenium, 100 µmol/l pargyline and the indicated concentrations of native LDL or mox-LDL. After 24 h of incubation, 50 µmol/L of 5HT was added and incubated for another 24 h. The experiment was terminated by aspiration of the medium, then the plates were washed with PBS, and 0.5 ml of 2% (w/v) crude pancreatic trypsin in Dulbecco’s PBS containing 152 mM EDTA was added to each dish. The dishes were incubated at room temperature for 2 min before the addition of 1.5 mL of serum. The contents of each dish were diluted to 20 ml with Isotone II (Coulter Electronics, Hialeah, Florida) and the cell number was determined using a coulter counter. Triplicate counts were taken for each plate and quadruplicates were used for each determination.

Statistical analysis.   Data are presented as mean ± standard error (SE) and expressed as percentage change from the baseline value for the unstimulated cells (=100%) except for cell number. Data were analyzed by one way analysis of variance (ANOVA) and the Bonferroni/Dunn test was used to identify differences among the groups when the overall F statistics were significant.


   Results
Top
 Abstract
 Materials and methods
 Results
Discussion
 References
 
Effects of LDL and mox-LDL on 3H-thymidine incorporation by VSMC.   The effect of increasing concentrations of LDL or mox-LDL on 3H-thymidine incorporation into DNA of VSMC is shown in Figure 1. Both LDL and mox-LDL induced significant proliferation of VSMCs ({approx}1.2- or 2-fold increase in thymidine incorporation, respectively, over the control; p < 0.05). In contrast to native LDL, mox-LDL at a concentration as low as 20 µg/ml induced a significant proliferation of VSMC. At concentrations greater than 20 µg/ml, mox-LDL had a significantly greater proliferative effect on VSMC when compared with native LDL. At high concentrations (>250 µg/mL) both LDL and mox-LDL were cytotoxic to VSMC (data not shown).



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  		Figure 1 Effect of LDL and mox-LDL on 3H-thymidine incorporation into VSMC. The amount of 3H-thymidine incorporation into DNA was determined in synchronized VSMCs stimulated with indicated concentrations of LDL or mox-LDL in serum free medium for 48 h. One hundred percent equals the baseline value for 3H-thymidine incorporation into DNA (268 ± 12 cpm). Values shown are mean ± standard error. LDL = low density lipoprotein; mox-LDL = mildly oxidized low density lipoprotein; VSMC = vascular smooth muscle cell. n = 24; *p < 0.01 compared with control; **p < 0.001 compared with control.

 
Effect of 5HT on VSMC preincubated with LDL.   The interaction between increasing concentrations of LDL and 5HT on 3H-thymidine incorporation into DNA of VSMC is shown in Figure 2. Serotonin alone at an added concentration of 5 µM and 50 µM induced 3H-thymidine incorporation by 188 ± 11% (259 ± 21 cpm to 478 ± 28 cpm) and 211 ± 11% (254 ± 21 cpm to 534 ± 28 cpm), respectively (p < 0.05 compared with the baseline value).



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  		Figure 2 Effect of 5HT on VSMC preincubated with LDL. Synchronized VSMCs were stimulated without or with different concentrations of LDL. After 24 h of incubation, indicated concentration of 5HT was added and incubated for an additional 24 h. One hundred percent equals the baseline value for 3H-thymidine incorporation into DNA (254 ± 21 cpm). Values shown are mean ± standard error. 5HT = serotonin. All other abbreviations as in Figure 1. n = 24; *p < 0.01 compared with corresponding control; **p < 0.01 compared with corresponding lipoprotein group.

 
Effect of 5HT on VSMC preincubated with mox-LDL.   The interaction between increasing concentrations of mox-LDL and 5HT on 3H-thymidine incorporation into DNA of VSMC is shown in Figure 3. In contrast to native LDL, in the presence of mox-LDL both the concentrations of 5HT tested (5 µM and 50 µM) significantly increased 3H-thymidine incorporation into VSMC when compared with the corresponding values with 5HT alone. Although mox-LDL (120 µg/ml) alone increased 3H-thymidine incorporation by 100% (280 ± 19 cpm to 567 ± 26 cpm), 5 µM and 50 µM 5HT alone induced 3H-thymidine incorporation by 100% (280 ± 19 cpm to 560 ± 14 cpm) and 140% (280 ± 19 cpm to 672 ± 14 cpm), respectively, over the control. However, the addition of 5 µM and 50 µM 5HT to VSMC preincubated with 120 µg/ml of mox-LDL resulted in a 306 ± 25% and a 453 ± 54% increase in 3H-thymidine incorporation over the control, respectively (Fig 3).



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  		Figure 3 Effect of 5HT on VSMC preincubated with mox-LDL. Synchronized VSMCs were stimulated with different concentrations of mox-LDL. After 24 h of incubation, indicated concentration of 5HT was added and incubated for an additional 24 h. One hundred percent equals the baseline value for 3H-thymidine incorporation into DNA (280 ± 19 cpm). Values shown are mean ± standard error. Abbreviations as in Figure 2. n = 24; *p < 0.01 compared with corresponding control; **p < 0.01 compared with corresponding lipoprotein group.

 
Effect of LDL or mox-LDL and 5HT on VSMC number.   To determine whether the induction of DNA synthesis by LDL, mox-LDL or 5HT resulted in an increase in cell number, we also examined the interaction between LDL or mox-LDL and 5HT on cell number of VSMC (Fig. 4). The results show that incubation of VSMC with LDL or mox-LDL, or 5HT alone, produced an increase in cell number compared with the baseline. When 5HT was added to LDL or mox-LDL pretreated cells, there was a further increase in the cell number.



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  		Figure 4 Effect of LDL or mox-LDL and 5HT on cell number. Vascular smooth muscle cells (5 x 104) were plated in 2 mL of serum free medium. After synchronization, vascular smooth muscle cells were stimulated with different concentrations of LDL or mox-LDL for 24 h, followed by incubation with 50 µmol/L of 5HT for 24 h. Then cells were trypsinized and counted as described in the Materials and Methods section. Data represent the mean ± SE from triplicate dishes from three different experiments. Baseline value is 256, 650 ± 13, 430 cells/plate. Abbreviations as in Figure 2. n = 12. *p < 0.01 compared with control; **p < 0.05 compared with corresponding control.

 
Effect of 5HT2 receptor antagonist (LY281067) on synergistic interaction between LDL, mox-LDL and 5HT.   We examined whether a 5HT2 receptor antagonist would inhibit the synergistic interaction between LDL, mox-LDL and 5HT on cell proliferation response. In a previous report (28) we have shown that LY281067, a 5HT2 receptor antagonist, completely blocked the 5HT induced 3H-thymidine incorporation without any cytotoxic effect. In this study, VSMCs were incubated with LDL or mox-LDL for 24 h and then 10 µg/ml of LY281067 was added to the media. Four hours after the addition of LY281067, 50 µM 5HT was added, and 24 h later 3H-thymidine incorporation was measured. At 10 µg/ml, LY281067 blocked only the proliferative effect of 5HT and its synergistic interaction with LDL or mox-LDL but not the mitogenic effect of LDL or mox-LDL (Fig. 5).



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  		Figure 5 Effect of 5HT2 receptor antagonist (LY281067) on 5HT induced proliferation of LDL or mox-LDL pretreated VSMC. Synchronized VSMCs were stimulated with different concentrations of LDL or mox-LDL. 10 µg/mL of LY281067 was added 4 h before the addition of 5HT. After 24 h of incubation with LDL or mox-LDL, 50 µmol/L of 5HT was added and incubated for an additional 24 h. 3H-thymidine (1 µCi/plate) was added during the last 5 h of incubation. One hundred percent equals the baseline value for 3H-thymidine incorporation into DNA (276 ± 17 cpm). Values shown are mean ± standard error. Abbreviations as in Table 2. n = 24; *p < 0.05 compared with LDL group control; **p < 0.05 compared with corresponding LDL and 5HT group; @p < 0.01 compared with mox-LDL group control; &p < 0.001 compared with 5HT; $p < 0.001 compared with corresponding mox-LDL and 5HT group.

 
Effect of pertussis toxin (PTX) on synergistic interaction between LDL, mox-LDL and 5HT.   To distinguish between the proliferative mechanisms of 5HT and LDL or mox-LDL, specific inhibitors of signal transduction pathways were used. Pertussis toxin, which ADP-ribosylates and inactivates susceptible Gi-subclass of G proteins, at a concentration of 10 ng/mL abolished the mitogenic effect of 5HT and its synergistic interaction with LDL and mox-LDL (Fig. 6). Pertussis toxin itself was not cytotoxic to the cells at the concentrations tested (data not shown). In contrast, PTX at the concentration of 10 ng/mL did not prevent the LDL or mox-LDL induced VSMC proliferation (Fig. 6).



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  		Figure 6 Effect of pertussis toxin (PTX) on 5HT induced proliferation of LDL or mox-LDL pretreated VSMC. Synchronized VSMCs were stimulated with different concentrations of LDL or mox-LDL in the presence or absence of 10 ng/mL of PTX. After 24 h of incubation with LDL or mox-LDL, 50 µmol/L of 5HT was added and incubated for an additional 24 h. One hundred percent equals the baseline value for 3H-thymidine incorporation into DNA (257 ± 21 cpm). Values shown are mean ± standard error. Abbreviations as in Figure 2. n = 24; *p < 0.05 compared with corresponding control; **p < 0.01 compared with LDL and 5HT group; @p < 0.01 compared with mox-LDL group control; &p < 0.001 compared with 5HT; $p < 0.001 compared with corresponding mox-LDL and 5HT group.

 
Effect of polyinosinic acid (poly-I) on synergistic interaction between LDL, mox-LDL and 5HT.   To evaluate the possible role of the scavenger receptor in the proliferative mechanisms of LDL or mox-LDL, poly-I, which binds to scavenger receptors, was used (37). Polyinosinic acid at concentration of 25 µg/mL did not inhibit the mitogenic effect of 5HT or LDL or mox-LDL (Fig. 7). Polyinosinic acid itself at this concentration was not cytotoxic to the cells at the concentrations tested (data not shown).



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  		Figure 7 Effect of poly-I on lipoprotein and 5HT induced VSMC proliferation. Synchronized VSMCs were stimulated with different concentrations of LDL or mox-LDL in the presence or absence of 25 µg/mL of poly-I. After 24 h of incubation with LDL or mox-LDL, 50 µmol/L of 5HT was added and incubated for an additional 24 h. One hundred percent equals the baseline value for 3H-thymidine incorporation into DNA (274 ± 18 cpm). Values shown are mean ± standard error. poly-I = polyinosinic acid. All other abbreviations as in Figure 2. n = 10; *p < 0.05 compared with corresponding control; **p < 0.05 compared with corresponding lipoprotein group.

 

   Discussion
Top
 Abstract
 Materials and methods
 Results
 Discussion
References
 
This study demonstrates an interaction between LDL or mox-LDL and 5HT on proliferation of VSMCs. Polyinosinic acid did not inhibit the proliferative effect of mox-LDL, suggesting that, unlike oxidized LDL, mox-LDL may not be recognized by the scavenger receptors. The LY281067 reversed only the mitogenic effect of 5HT but not that of LDL or mox-LDL on VSMCs. In general, 5HT-mediated responses are known to be PTX sensitive, which indicates that serotonergic receptors are coupled with the Gi subfamily of the G proteins alpha-subunit which catalyzes the hydrolysis of phosphoinositide biphosphate. Similarly, PTX reversed the mitogenic effect of 5HT but did not inhibit the effect of LDL or mox-LDL on VSMCs.

Effect of LDL and mox-LDL on VSMC proliferation.   The growth promoting effects of native or oxidized LDL on VSMCs are conflicting (6–8,21,22,38–40). Although LDL has been reported to be cytotoxic to VSMCs in some studies (12), others report growth stimulatory effects of LDL on VSMCs in combination with serum (4–6) or lipoprotein-depleted serum (8,25). In the studies involving serum containing media, lipoproteins may have potentiated the effects of growth factors present in the serum. However, others have reported that LDL itself is a mitogen for vascular endothelial or VSMCs under serum-free medium conditions (8,38). The discrepancies in the results may be due to the source or the way SMCs were isolated. In this study, we used explant-derived SMCs which may be phenotypically different from those isolated by enzymatic methods. As reported by others (10,13,14,41), in our study, both LDL and mox-LDL exhibited a biphasic effect on VSMCs: a proliferative effect at low concentrations (20–120 µg protein/ml) and a cytotoxic effect at high doses (>200 µg protein/ml). It has been demonstrated that VSMCs can modify LDL in vitro. The increased mitogenic effect of mox-LDL or oxidized LDL is attributed to the chemical changes brought about by the oxidation processes to the LDL components such as, generation of hydrogen peroxide and conversion of phosphatydilcholine to lysophosphatidylcholine (Lyso-PC). During the early periods of oxidation, there is a significant accumulation of peroxides and presumably other reactive oxygen intermediates, whereas the Lyso-PC levels are still relatively low. After 24 h of oxidative stress, LDL contains large amounts of Lyso-PC but only small amounts of reactive oxygen intermediates (42). Extensive oxidation is also associated with the formation of several oxysterols (42). Though oxygen radicals are generally considered cytotoxic (43,44), recent studies have demonstrated that, at low concentrations, oxygen radicals may affect several of the mechanisms involved in cell growth (45). Like most of the growth factors, they induce intracellular alkalization and active transcription of early response genes (46–48). There is also evidence that intracellular oxygen radicals play a direct role in regulation of gene transcription (49,50). At lower concentrations, Lyso-PC also acts as a mitogen for VSMC (51). The growth inhibitory effect of mox-LDL at higher concentrations may be due to the higher levels of reactive oxygen species, Lyso-PC or cytotoxic concentrations of oxysterols, or combination of all three. It is possible that some degree of oxidation of LDL may have occurred in our experiments due to incubation with VSMCs for 48 h. To exclude this, we repeated the experiments in the presence of butylated hydroxy toluene, vitamin E, vitamin C and N-acetyl cystine and found that there was no change in the effect of LDL on SMC proliferation (data not shown) indicating that the mitogenic effect of LDL in this study was not due to mild oxidation of LDL during culture.

Role of LDL receptors in mox-LDL induced VSMC proliferation.   The mechanism for the growth-promoting activity of LDL or mox-LDL is unknown. New membrane components, notably cholesterol and phospholipids, are required for cell division. When cells are grown in serum-free medium, LDL is internalized by the cell after it is bound to the LDL receptor. Lysosomal degradation of this internalized LDL provides the cell with unesterified cholesterol for use in membrane synthesis. Conversely, Bjorkerud suggested that the growth promoting effects were specific for the lipoproteins (8). It is assumed that both the protein and lipid components of lipoprotein are essential for proliferation of VSMC (9,10). Previous studies suggest that growth promoting effects of native LDL require the presence of LDL receptors. Because both LDL and mox-LDL are taken up by LDL B/E receptors (41), it is possible that LDL receptors of VSMCs may mediate the uptake of both LDL and mox-LDL into VSMCs. Similarly, poly-I, which binds to the alternate scavenger receptors, did not inhibit the proliferative effect of LDL or mox-LDL.

Effect of mox-LDL and 5HT on VSMC proliferation.   Generally, serotonergic receptors are found to be coupled to pertussis toxin sensitive G-proteins (26,52). In this study, pertussis toxin could only reverse the mitogenic effect of 5HT and its synergistic effect with LDL without affecting the mitogenic effect of LDL. Previous studies have indicated that Lyso-PC mediated mitogen activated protein kinase (MAP-kinase) stimulation is insensitive to PTX (51). In this study PTX also failed to reverse the mitogenic effect of mox-LDL indicating that mox-LDL mediated mitogenic effects may not be mediated via the G1-Ras/Raf pathway. A MAP-kinase independent pathway shown to be activated by cellular stress, tumor necrosis factor and ultraviolet light (a form of oxidative stress) is represented by stress activated protein kinases, a newly described family of serine/threonine kinases termed as c-Jun N-terminal kinases/stress activated kinase (JNK/SAPK) (53–55). Like mitogen activated protein kinases, JNK/SAPK have been known to activate AP-1 (activator protein-1) inducing activity through c-Jun (56,57). It has been recently reported that both Lyso-PC and hydrogen peroxide, the major components of oxidized LDL, stimulate JNK (51,58). Therefore, the activation of a MAP pathway by 5HT and redoxisensitive or JNK/SAPK pathway by mox-LDL may explain the synergistic interaction in our studies. Thus, at sites of vascular injury, platelet derived 5HT may interact with the mox-LDL in stimulating the VSMC proliferation.

Conclusions.   In the formation of atherosclerotic plaques, deposits of LDL in the artery wall and platelet aggregation are important steps. Low density lipoprotein (23,30,32), mox-LDL (33) and oxidized LDL (59) induce platelet aggregation and increase the release of 5HT from platelets (25,31–33). Seewald et al. (60) have demonstrated that LDL and thrombin act synergistically, and we have demonstrated that TXA2 and serotonin (28) act synergistically in stimulating SMC proliferation. Thus, it is conceivable that, in addition to other classical growth factors such as platelet derived growth factor, 5HT alone or in combination with LDL or mox-LDL may also contribute to the formation of plaque.


   Acknowledgments
 
We thank Shirley McWhorter for assisting in typing the manuscript.


   Footnotes
 
Dr. Benedict was supported by the National Institutes of Health, National Heart, Lung, and Blood Institute (grant RO1-HL39916) American Heart Association Grant-In-Aid.


   References
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 Abstract
 Materials and methods
 Results
 Discussion
 References
 

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