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
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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