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PCSK9 Induces Rat Smooth Muscle Cell Proliferation and Counteracts the Pleiotropic Effects of Simvastatin

Lupo, Maria Giovanna, Marchianò, Silvia, Adorni, Maria Pia, Zimetti, Francesca, Ruscica, Massimiliano, Greco, Maria Francesca, Corsini, Alberto, Ferri, Nicola·2021·International Journal of Molecular Sciences
DOI 10.3390/ijms22084114PMC PMC8073479
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Abstract

Human atherosclerotic plaque contains smooth muscle cells (SMCs) negative for the contractile phenotype (α-smooth muscle actin) but positive for proprotein convertase subtilisin/kexin type 9 (PCSK9). Thus, we generated rat SMCs which overexpressed human PCSK9 (SMCsPCSK9) with the aim of investigating the role of PCSK9 in the phenotype of SMCs. PCSK9 overexpression in SMCsPCSK9 led to a significant downregulation of the low-density lipoprotein receptor (Ldlr) as well as transgelin (Sm22α), a marker of the contractile phenotype. The cell proliferation rate of SMCsPCSK9 was significantly faster than that of the control SMCs (SMCspuro). Interestingly, overexpression of PCSK9 did not impact the migratory capacity of SMCs in response to 10% FCS, as determined by Boyden’s chamber assay. Expression and activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (Hmgcr) was significantly increased in the presence of PCSK9, both in SMCPCSK9 and after treatment with recombinant PCSK9. The transcriptional activity of sterol regulatory element-binding protein (SREBP) was also increased in the presence of PSCK9, suggesting a direct role of PCSK9 in the control of SRE-responsive genes, like HMGCR. We also observed that cholesterol biosynthesis is elevated in SMCPCSK9, potentially explaining the increased proliferation observed in these cells. Finally, concentration-dependent experiments with simvastatin demonstrated that SMCsPCSK9 were partially resistant to the antiproliferative and antimigratory effect of this drug. Taken together, these data further support a direct role of PCSK9 in proliferation, migration, and phenotypic changes in SMCs—pivotal features of atherosclerotic plaque development. We also provide new evidence on the role of PCSK9 in the pharmacological response to statins—gold standard lipid-lowering drugs with pleiotropic action.

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Figure 1 Effect of PCSK9 overexpression on Ldlr in SMCs. SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (3 × 105 cells/well in a 6-well tray). The day after, the medium was changed with DMEM containing either a high (10%) or low (0.4%) concentration of FCS and, after an additional 72 h, the total cell lysates were prepared. (A) PCSK9 and Ldlr protein expression were evaluated by Western blot analysis. α-tubulin was used as the loading control. Panel A shows representative images of three independent experiments. (B) Densitometric readings for the Ldlr were evaluated using ImageLab software. Ldlr, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SMCPCSK9, smooth muscle cells overexpressing PCSK9. Differences between conditions were assessed by Student’s t-test and one-way ANOVA (when necessary). * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 1 Effect of PCSK9 overexpression on Ldlr in SMCs. SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (3 × 105 cells/well in a 6-well tray). The day after, the medium was changed with DMEM containing either a high (10%) or low (0.4%) concentration of FCS and, after an additional 72 h, the total cell lysates were prepared. (A) PCSK9 and Ldlr protein expression were evaluated by Western blot analysis. α-tubulin was used as the loading control. Panel A shows representative images of three independent experiments. (B) Densitometric readings for the Ldlr were evaluated using ImageLab software. Ldlr, low-density lipoprotein receptor; PCSK9, proprotein convertase subtilisin/kexin type 9; SMCPCSK9, smooth muscle cells overexpressing PCSK9. Differences between conditions were assessed by Student’s t-test and one-way ANOVA (when necessary). * p < 0.05; ** p < 0.01; *** p < 0.001.CC BY · PubMed Central
Figure 2 Effect of PCSK9 overexpression on SMC proliferation and migration. (A) SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (2 × 104 cells/well in a 24-well tray). Cell number was estimated by Sulforhodamine B staining after 24 h (day 0) and at day 2, 5, and 9. The data are expressed as mean ± SD of absorbance at 570 nm. Error bars (not shown) are within the symbol limits. For each time point, *** p < 0.001 SMCspuro vs. SMCsPCSK9. (B) Representative images of SMCpuro and SMCPCSK9 after 9 days of growth. (C) SMCpuro and SMCPCSK9 were cultured for 24 h in DMEM containing 0.4% FCS. After 24 h, cells were harvested by trypsinization and migration was measured by Boyden’s chamber chemotactic assay, using FCS as the chemotactic agent at the indicated amount. Transmigrated cells were counted after 6 h of migration in six random high-power fields (HPFs) under high magnification (objective lens 20×). For each cell type, *** p < 0.001 10% vs. 0.4% FCS. (D) SMCpuro and SMCPCSK9 were cultured in the presence of low (0.4%) and high (10%) FCS concentrations. After 24 h, total RNA was prepared and the mRNA level of Sm22α was determined by quantitative real-time PCR. * p < 0.05 and *** p < 0.001. Differences between conditions were assessed by Student’s t-test and one-way ANOVA (when necessary).
Figure 2 Effect of PCSK9 overexpression on SMC proliferation and migration. (A) SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (2 × 104 cells/well in a 24-well tray). Cell number was estimated by Sulforhodamine B staining after 24 h (day 0) and at day 2, 5, and 9. The data are expressed as mean ± SD of absorbance at 570 nm. Error bars (not shown) are within the symbol limits. For each time point, *** p < 0.001 SMCspuro vs. SMCsPCSK9. (B) Representative images of SMCpuro and SMCPCSK9 after 9 days of growth. (C) SMCpuro and SMCPCSK9 were cultured for 24 h in DMEM containing 0.4% FCS. After 24 h, cells were harvested by trypsinization and migration was measured by Boyden’s chamber chemotactic assay, using FCS as the chemotactic agent at the indicated amount. Transmigrated cells were counted after 6 h of migration in six random high-power fields (HPFs) under high magnification (objective lens 20×). For each cell type, *** p < 0.001 10% vs. 0.4% FCS. (D) SMCpuro and SMCPCSK9 were cultured in the presence of low (0.4%) and high (10%) FCS concentrations. After 24 h, total RNA was prepared and the mRNA level of Sm22α was determined by quantitative real-time PCR. * p < 0.05 and *** p < 0.001. Differences between conditions were assessed by Student’s t-test and one-way ANOVA (when necessary).CC BY · PubMed Central
Figure 3 PCSK9 induced Hmgcr and cholesterol biosynthesis. SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (3 × 105 cells/well in a 6-well tray). The day after, the medium was changed with DMEM containing either high (10%) or low (0.4%) FCS concentration and, after an additional 72 h, the total cell lysates were prepared. (A) Hmgcr expression was evaluated by Western blot analysis. α-tubulin was used as the loading control. (B) Bar graphs of the quantification of Western blot analysis. (C) SMCs were cultured in DMEM containing 10% FCS in the presence or absence of 5 µg/mL of human recombinant PCSK9 (recPCSK9) for 24 h and 48 h. At the end of the incubation, the total protein extracts were prepared, and Hmgcr levels were determined by Western blot analysis. GAPDH was used as the loading control. (D) Bar graphs of quantification of Western blot analysis. (E) SMCpuro and SMCPCSK9 were cultured in DMEM/10% FCS containing [2-14C]-acetate for 48 h. [2-14C]-acetate incorporation into cellular sterols was used to assay cholesterol biosynthesis. Each point represents the mean ± SD of triplicate dishes. (F) SMCpuro and SMCPCSK9 were transfected with pGL3-PCSK9-D4. Forty-eight hours post-transfection, the medium was replaced with DMEM containing 10% FCS, and, after an additional 24 h, luciferase activities were determined by Neolite reagent. Luciferase activities were normalized to the β-galactosidase activity of the cotransfected pCMV-β construct. Differences between conditions were assessed by Student’s t-test and one-way ANOVA (when necessary). * p < 0.05; ** p < 0.01; *** p < 0.001.
Figure 3 PCSK9 induced Hmgcr and cholesterol biosynthesis. SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (3 × 105 cells/well in a 6-well tray). The day after, the medium was changed with DMEM containing either high (10%) or low (0.4%) FCS concentration and, after an additional 72 h, the total cell lysates were prepared. (A) Hmgcr expression was evaluated by Western blot analysis. α-tubulin was used as the loading control. (B) Bar graphs of the quantification of Western blot analysis. (C) SMCs were cultured in DMEM containing 10% FCS in the presence or absence of 5 µg/mL of human recombinant PCSK9 (recPCSK9) for 24 h and 48 h. At the end of the incubation, the total protein extracts were prepared, and Hmgcr levels were determined by Western blot analysis. GAPDH was used as the loading control. (D) Bar graphs of quantification of Western blot analysis. (E) SMCpuro and SMCPCSK9 were cultured in DMEM/10% FCS containing [2-14C]-acetate for 48 h. [2-14C]-acetate incorporation into cellular sterols was used to assay cholesterol biosynthesis. Each point represents the mean ± SD of triplicate dishes. (F) SMCpuro and SMCPCSK9 were transfected with pGL3-PCSK9-D4. Forty-eight hours post-transfection, the medium was replaced with DMEM containing 10% FCS, and, after an additional 24 h, luciferase activities were determined by Neolite reagent. Luciferase activities were normalized to the β-galactosidase activity of the cotransfected pCMV-β construct. Differences between conditions were assessed by Student’s t-test and one-way ANOVA (when necessary). * p < 0.05; ** p < 0.01; *** p < 0.001.CC BY · PubMed Central
Figure 4 PCSK9-overexpressing cells were partially resistant to simvastatin action. (A) SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (2 × 104 cells/well in a 24-well tray). After 24 h, cells were incubated in the presence or absence of the indicated concentration of simvastatin and, in a separate well, the cell number was estimated by Sulforhodamine B (SRB) staining (at time 0). After 72 h of incubation, the cell number was evaluated by SRB and cell proliferation was determined. (B) Representative images of SMCpuro and SMCPCSK9 after 72 h of incubation with 1.5 µM simvastatin in DMEM/10% FCS. (C) SMCpuro and SMCPCSK9 were cultured for 24 h in DMEM containing 0.4% FCS. After 24 h, cells were harvested by trypsinization and incubated in the presence or absence of the indicated concentration of simvastatin during 6 h of migration, measured by Boyden’s chamber chemotactic assay. FCS (10%) was used as the chemotactic agent. Transmigrated cells were counted after migration in six random high-power fields (HPFs) under high magnification (objective lens 20×). Differences between conditions were assessed by Student’s t-test. ** p < 0.01; *** p < 0.001 SMCPCSK9 vs. SMCpuro.
Figure 4 PCSK9-overexpressing cells were partially resistant to simvastatin action. (A) SMCpuro and SMCPCSK9 were seeded in DMEM/10% FCS (2 × 104 cells/well in a 24-well tray). After 24 h, cells were incubated in the presence or absence of the indicated concentration of simvastatin and, in a separate well, the cell number was estimated by Sulforhodamine B (SRB) staining (at time 0). After 72 h of incubation, the cell number was evaluated by SRB and cell proliferation was determined. (B) Representative images of SMCpuro and SMCPCSK9 after 72 h of incubation with 1.5 µM simvastatin in DMEM/10% FCS. (C) SMCpuro and SMCPCSK9 were cultured for 24 h in DMEM containing 0.4% FCS. After 24 h, cells were harvested by trypsinization and incubated in the presence or absence of the indicated concentration of simvastatin during 6 h of migration, measured by Boyden’s chamber chemotactic assay. FCS (10%) was used as the chemotactic agent. Transmigrated cells were counted after migration in six random high-power fields (HPFs) under high magnification (objective lens 20×). Differences between conditions were assessed by Student’s t-test. ** p < 0.01; *** p < 0.001 SMCPCSK9 vs. SMCpuro.CC BY · PubMed Central

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Authors
Lupo, M. G., Marchianò, S., Adorni, M. P., Zimetti, F., Ruscica, M., Greco, M. F., Corsini, A., & Ferri, N.
Affiliations
  • Dipartimento di Scienze del Farmaco, Università degli Studi di Padova, 35131 Padova, Italy
  • Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA 98195, USA
  • Unit of Neurosciences, Department of Medicine and Surgery, Università degli Studi di Parma, 43125 Parma, Italy
  • Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, 43124 Parma, Italy
  • Dipartimento di Scienze Farmacologiche e Biomolecolari, Università degli Studi di Milano, 20133 Milan, Italy
ORCID
Venue
International Journal of Molecular Sciences
Publisher
MDPI AG
Published
2021-04-16
Volume
22
Issue
8
Pages
4114
ISSN
1422-0067
DOI
10.3390/ijms22084114
PMC
PMC8073479
Cited by
15
References
41
Language
en
Licence
creativecommons.org/licenses/by/4.0/ (vor)

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Lupo, M. G., Marchianò, S., Adorni, M. P., Zimetti, F., Ruscica, M., Greco, M. F., Corsini, A., & Ferri, N. (2021). PCSK9 Induces Rat Smooth Muscle Cell Proliferation and Counteracts the Pleiotropic Effects of Simvastatin. International Journal of Molecular Sciences. https://doi.org/10.3390/ijms22084114

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