PCSK9 knock-out mice are protected from neointimal formation in response to perivascular carotid collar placement

Background and aims: Proprotein convertase subtilisin kexin type 9 (PCSK9) induces degradation of the low-density lipoprotein-receptor (LDLR). Smooth muscle cells (SMCs) in human atherosclerotic plaques and cultured SMCs express PCSK9. The present study aimed at defining the role of PCSK9 on vascular response to injury. Methods: Carotid neointimal lesions were induced by positioning a non-occlusive collar in PCSK9 knockout (PCSK9(-/-)) and wild type littermate (PCSK9(+/+)) mice. Results: In PCSK9(-/-) mice, we observed a significantly less intimal thickening (p < 0.05), a lower intimal media ratio (p < 0.02), and a tendency to higher lumen area, compared to PCSK9(+/+) mice. When compared with PCSK9(-/-), lesions of PCSK9(+/+) mice had a higher content of SMCs (p < 0.05) and collagen (p < 0.05), while no difference was observed in the accumulation of macrophages. PCSK9 was detectable in both left and right carotids artery in regions occupied by medial and neointimal SMCs. SMCs freshly isolated from PCSK9(-/-), when compared to PCSK9(+/+) cells, showed higher levels of alpha-smooth muscle actin (alpha-SMA; 2.24 +/- 0.36 fold; p < 0.01) and myosin heavy chain II (MHC-II; 8.65 +/- 1.55 fold; p < 0.01), and lower levels of caldesmon mRNA (-54 +/- 14%; p < 0.01). PCSK9(-/-) cells also showed a slower proliferation rate, and an impaired migratory capacity and G1/S progression of the cell cycle. The reconstitution of PCSK9 expression, by retroviral infection of PCSK9(-/-) SMCs, led to a downregulation of a-SMA (-56 +/- 2%; p < 0.01), MHC-II(-45% +/- 25.5 fold: p = 0.06) and calponin (-25% +/- 0.8 fold: p < 0.05) and induction of caldesmon mRNA (1.46 +/- 0.3 fold; p < 0.05). Proliferation rate of SMCs PCSK9(-/-) was significantly lower compared to PCSK9 reconstituted cells. Conclusions: Taken together, the present results suggest that PCSK9, by sustaining SMC synthetic phenotype, proliferation, and migration, may play a pro-atherogenic role in the arterial wall

Genotypic [n(%)] and allelic frequencies (%) of the rs9370867 SNP in the PLIC population.

<p>Genotypic [n(%)] and allelic frequencies (%) of the rs9370867 SNP in the PLIC population.</p

mRNA expression of LDL-R and IDOL and LDL uptake in macrophages from N342 and S342 macrophages.

<p>Panel A shows mRNA expression for LDL-receptor and for IDOL in macrophages obtained from N342 and S342 carriers (n = 5 for both genotypes) cultured in complete RPMI medium The results, normalized for the expression of an housekeeping gene (18S), data are shown as mean ± SEM. Next flow cytometry was used to study the LDL uptake in macrophages obtained from N342 or S342 carriers. To determine the LDL uptake, macrophages were incubated with fluorescently labelled LDL (DiO-LDL, 10 μg/mL) for 2 hours at 37°C. Panel B shows representative images for flow cytometry in N342 and S342 macrophages, with the gating strategy of macrophages (inside panel) and the fluorescence intensity for Dio-LDL while panel C shows the results from five N342 and five S342 patients (mean intensity fluorescence is given, mean ± SEM is shown).</p

Incidence of coronary heart disease (CHD), peripheral artery disease (PAD) and cardiovascular events (CVE) in the PLIC population according to the rs9370867 SNP.

<p>Panels A, B and C shows the incidence (%) of CHD, PAD and CVE for GG, AG or AA carriers of the rs9370867 IDOL SNP. Panel D shows the risk CHD, PAD and CVE for the carriers of A allele of the rs9370867 vs carriers of the G allele. Odds Ratios (OR, the 95% Confidence Interval (C.I.), adjusted for age, gender, lipid profile, systolic blood pressure, glucose levels and therapies) are not statistically significant.</p

Anthropometric and biochemical characteristics according to the rs9370867 SNP in subjects from the PLIC population.

<p>Anthropometric and biochemical characteristics according to the rs9370867 SNP in subjects from the PLIC population.</p

Heart left ventricular mass and carotid intima media thickness of the common carotid arteries in the PLIC population according to the rs9370867 IDOL SNP.

<p>Panel A shows the mean ± SD for left ventricular mass for GG, AG or AA carriers. Panel B shows the mean ± SD of carotid intima media thickness of the common carotid arteries (right and left) for GG, AG or AA carriers.</p

Class II PI3Ks are involved in HDL₃-dependent EC migration.

<p>Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and HDL₃-mediated cell migration was determined by Transwell assays as above. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 4 (A), 5 (B) and 5 (C) independent experiments. *p<0.05; **p<0.01.</p

Class II PI3Ks do not regulate Akt or ERK activation.

<p>HUVEC transfected with the indicated siRNAs were serum starved in M199 containing 0.5% FBS overnight before stimulation with HDL₃ or S1P for 10 min. Akt and ERK phosphorylation was assessed by Western blotting. Densitometry analysis shows means ± SEM from 3–4 (Akt) and 4 (ERK) independent experiments.</p

Class II PI3Ks are involved in S1P-dependent EC migration.

<p>(A) Expression levels of PI3K-C2α and PI3K-C2β in HUVEC transfected with a scrambled siRNA or siRNAs (“Sequences 1”) specifically targeting the indicated enzymes. (B-D) Twenty four hours after transfection with the indicated siRNAs cells were serum starved overnight and S1P-mediated cell migration was determined by Transwell assays as above. Data are expressed as percentage of migration of cells transfected with scrambled siRNA and unstimulated (control) and are means ± SEM from 17 (B), 11 (C) and 6 (D) independent experiments. *p<0.05; **p<0.001.</p

Effect of PI3Ks downregulation on EC apoptosis.

<p>(A) Results from caspase 3 assay performed on lysates from HUVEC obtained 48 h after transfection with the indicated siRNAs. Data are means ± SEM from 3–4 independent experiments. Student’s t-Test: un-paired *p<0.05 vs cells transfected with PI3K-C2β siRNA. (B) HUVEC were transfected with the indicated siRNAs. After 48 h the percentage of apoptotic cells was determined by Annexin V staining. Annexin V positive and propidium iodide negative cells were gated. Results are expressed as means ± SEM from 3 independent experiments. *p<0.05 vs scrambled siRNA treated cells.</p