1. Phosphatidylinositol 3-Kinase and Calcium-Activated Transcription Pathways Are Required for VLDL-Induced Smooth Muscle Cell Proliferation
by Larissa Lipskaia, Marie-Luce Pourci, Claudine Deloménie, Laurent Combettes, Dominique Goudounèche, Jean-Louis Paul, Thierry Capiod, and Anne-Marie Lompré
Circulation Research
Volume 92(10):
May 30, 2003
Copyright © American Heart Association, Inc. All rights reserved.

2. Figure 1. Effect of VLDL on proliferation of SMCs
Figure 1. Effect of VLDL on proliferation of SMCs. A, SMCs were treated for 48 hours either with 0.1% LPDS in the presence of indicated concentrations of VLDL or HDL or with 10% FCS. B, SMCs were treated for 5 to 96 hours with VLDL (0.15 g/L proteins=1 mmol/L triglycerides).
Figure 1. Effect of VLDL on proliferation of SMCs. A, SMCs were treated for 48 hours either with 0.1% LPDS in the presence of indicated concentrations of VLDL or HDL or with 10% FCS. B, SMCs were treated for 5 to 96 hours with VLDL (0.15 g/L proteins=1 mmol/L triglycerides). Each point represents the mean of 3 to 6 independent experiments. For each time point or each lipoprotein concentration, the number of cells was normalized to the number of cells in 0.1% LPDS with PBS as vehicle. Values were plotted as the percentage of change from control. *P<0.05 vs control.
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.

3. Figure 2. Effect of VLDL on caffeine- and ATP-evoked Ca2+ transients.
Figure 2. Effect of VLDL on caffeine- and ATP-evoked Ca2+ transients. A, Top, Caffeine-induced (10 mmol/L) or ATP-induced (10 μmol/L) increase in cytosolic [Ca2+] in control conditions. Middle, Caffeine-induced Ca2+ release in cells perfused with VLDL (1 mmol/triglycerides) (middle). Bottom, Caffeine- or ATP-induced Ca2+ release in cells pretreated for 4 hours with VLDL (1 mmol/L triglycerides). B, ATP-evoked Ca2+ transient in control conditions and in presence of VLDL (1 mmol/L triglycerides) for 4 hours. When applying the agonists, cells were perfused with an external solution containing no calcium and 100 μmol/L EGTA. C, Ca2+ influx generated by KCl depolarization in the presence of VLDL (1 mmol/L triglycerides) (top) is inhibited by diltiazem (60 μmol/L) (bottom).
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.

4. Figure 3. CREB phosphorylation is decreased in VLDL-treated SMCs
Figure 3. CREB phosphorylation is decreased in VLDL-treated SMCs. A, Immunofluorescence with a-pCREB in control conditions and after exposure to VLDL (1 mmol/L triglycerides) for 4 or 24 hours.
Figure 3. CREB phosphorylation is decreased in VLDL-treated SMCs. A, Immunofluorescence with a-pCREB in control conditions and after exposure to VLDL (1 mmol/L triglycerides) for 4 or 24 hours. Nuclei are visualized with Hoescht. Bar=10 μm. B, Western blot analysis of pCREB levels in control conditions and after exposure to VLDL (1 mmol/L triglycerides) for 4, 24, or 48 hours or to 10% FCS for 48 hours. After exposure, the membrane was stripped and probed with a-CREB. The films were scanned, and values of pCREB in each sample were normalized to the level of CREB. The results in the bar graph are presented as percent of control value and represent the average of 2 independent experiments. The bars refer to the lanes in the Western blot above. C, Western blot analysis of p53 expression in control condition or after exposure to VLDL for 4 or 48 hours.
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.

5. Figure 4. VLDLs activate the NFAT signaling pathway.
Figure 4. VLDLs activate the NFAT signaling pathway. A, RT-PCR analysis of NFAT mRNA isoforms in quiescent and proliferating SMCs. NFATc1 (NFATc), 1; NFATc2 (NFATp), 2; NFAT C4 (NFAT3), 3; NFAT C3 (NFAT4), 4. B, Immunofluorescence with a-NFATc3 in control conditions (a) and after 24 hours exposure to VLDL (1 mmol/L triglycerides) alone (b) or in presence of 5 μmol/L cyclosporin A (c) or 10 μmol/L LY (d). Bar=10 μm. C, Western blot analysis of the presence of NFAT in nuclear extracts from SMCs in control conditions (0.1% LPDS) or treated for 4 and 24 hours with VLDL (1 mmol/L triglycerides). Cells were also cultured for 24 hours with VLDL plus cyclosporin A (5 μmol/L) and with 10% FCS.
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.

6. Figure 5. Pharmacological analysis of the calcium signaling pathways of VLDL-induced proliferation.
Figure 5. Pharmacological analysis of the calcium signaling pathways of VLDL-induced proliferation. SMCs were cultured for 36 hours in the presence of 0.1% LPDS (vehicle) and VLDL (1 mmol/L triglycerides) alone (control) or with several drugs at indicated concentrations. Rya indicates ryanodine; Dil, diltiazem; LY, LY294002; and Rap, rapamycin. Data are expressed as percent of the cell number in control wells. The results represent the average of at least 3 independent experiments performed in pentaplicate. *P=0.05; **P<0.005; and ***P< vs control.
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.

7. Figure 6. VLDLs activate the PI3K-dependent signaling cascade.
Figure 6. VLDLs activate the PI3K-dependent signaling cascade. Western blot analysis of total extracts from SMCs in control conditions (0.1% LPDS) or treated for 30 minutes, 4 hours, and 24 hours with VLDL (1 mmol/L triglycerides) alone or in the presence of LY (10 μmol/L). The membranes were probed with a-pAkt (Thr308), a-pAkt (Ser473), a-pGSK-3β (Ser9), or a- p-FKHR (Ser256). Then they were stripped and probed with a-Akt, a-pPTEN (Ser380), and a-pPDK (Ser241), confirming equivalent protein loading.
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.

8. Figure 7. Schematic representation of VLDL-induced signaling pathway in vascular SMCs. VLDLR indicates VLDL receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homologue; PDK1/2-3, phosphoinositide-dependent protein kinase-1/2; Akt/PKB, protein kinase B; GSK-3β, glycogen synthase kinase-3β; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; IP3R, IP3 receptor; SOC, store-operated calcium channel; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; Dil, diltiazem, and Rya, ryanodine.
Figure 7. Schematic representation of VLDL-induced signaling pathway in vascular SMCs. VLDLR indicates VLDL receptor; PIP2, phosphatidylinositol 4,5-bisphosphate; PIP3, phosphatidylinositol 3,4,5-triphosphate; PTEN, phosphatase and tensin homologue; PDK1/2-3, phosphoinositide-dependent protein kinase-1/2; Akt/PKB, protein kinase B; GSK-3β, glycogen synthase kinase-3β; PLC, phospholipase C; IP3, inositol 1,4,5-triphosphate; IP3R, IP3 receptor; SOC, store-operated calcium channel; SERCA, sarco/endoplasmic reticulum Ca2+-ATPase; Dil, diltiazem, and Rya, ryanodine.
Larissa Lipskaia et al. Circ Res. 2003;92:
Copyright © American Heart Association, Inc. All rights reserved.