Fig 2: A: The levels of PCSK9 in mPCSK9 and LDLs from Ldlr-/-, LDb, and LTp mice. The recombinant mPCSK9 (50 ng per lane; Abcam) and LDLs (15 µg per lane) were separated using 4–20% SDS gel electrophoresis, followed by Western blot analysis using anti-PCSK9 (Biolegend; 1:3,000 dilution). The bands of PCSK9 in recombinant mPCSK9 and LDLs from Ldlr-/- and LDb mice are shown. LTp-LDL from LTp mice does not have any detectable PCSK9. mPCSK9 (Abcam) has a His-tag in the N terminal and the protein is glycosylated, as indicated by the manufacturer. The expected apparent molecular mass is 66 KDa. B: The effects of mPCSK9 and LDL from Ldlr-/-, LTp, and LDb mice on proatherogenic and autophagy gene expression in MCECs. MCECs were plated onto 12-well plates in duplicate. The next day each well was incubated with the indicated molecules (20 µg/ml) for 24 h. Cells were collected to extract RNA. mRNA levels were measured by real-time quantitative RT-PCR and normalized with ß-actin. The results are expressed as RQ (mean ± SD). We used two-way ANOVA to compare the effect of each factor (PBS, mPCSK9, Ldlr-/-LDL, LTp-LDL, and LDb-LDL) on all genes studied as a whole. The P of each comparison is listed on the figure. The Ps of individual gene effects are shown in Table 3. C: The addition of active mPCSK9 to LDb-LDL or LTp-LDL does not yield any synergistic effects on gene expression in MCECs. MCECs were plated onto 12-well plates in duplicate. The next day, each well was incubated with the indicated molecules (20 µg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. mRNA levels were measured by real-time quantitative RT-PCR and normalized with ß-actin. The results are expressed as RQ (mean ± SEM). Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). ns, not significant.

Fig 3: A: PCSK9 is associated with VLDL and LDL from Ldlr-/- and LDb mice. We used FPLC to separate plasma of Ldlr-/-, LTp, and LDb mice into lipoproteins. VLDL, LDL, HDL. VLDL and LDL fractions were pooled and concentrated using the Ultrafree 15 centrifugal filter device. We applied 20 µg of proteins to 4–20% SDS-PAGE. PCSK9 was detected by Western blot analysis using anti-PCSK9 (Biolegend; 1:3,000 dilution). The positions of PCSK9 are indicated. There is no detectable PCSK9 in LTp mice. B: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 µg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 µg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with ß-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). C: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 µg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 µg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with ß-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tail unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). D: LDb-LDL stimulated the gene expression levels of pro-atherosclerosis molecules on aortic primary ECs. Aortic primary ECs were cultured from C57BL/6J (C57_EC) and LDb (LDb_EC) mice and plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 µg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 µg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with ß-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). E: The comparison of binding and uptake of LDb-LDL and LTp-LDL to MCECs. Control DiI, DiI-LDb-LDL, or DiI-LTp-LDL was incubated with MCECs to study the binding and uptake of LDLs to MCECs. The detailed methods are described in the Materials and Methods. Binding of LDLs to MCECs was examined using a confocal microscope. The red dots represent the binding of LDLs to ECs. Both DiI-LDb-LDL and DiI-LTp-LDL bound to ECs after incubation at 4°C for 30 min. The uptake of LDLs to MCECs was carried out at 37°C for 4 h. There were strong uptakes of both LDLs to MCECs. There was no difference in the number of cells taken up by MCECs as determined by flow cytometry (LDb-LDL vs. LTp-LDL; 58 ± 3.2 vs. 56 ± 3.7). ns, not significant. F: LDb-LDL stimulated gene expression levels of pro-atherosclerosis molecules in MCECs. MCECs were plated onto 6-well plates in duplicate. The next day, each well was incubated with PBS, LDb-LDL (LDL from LDb mice associated with PCSK9, 20 µg/ml), or LTp-LDL (LDL from LTp mice without PCSK9, 20 µg/ml) for 24 h. Cells were collected to extract RNA. The experiment was performed three times. The mRNA levels were measured by real-time quantitative RT-PCR and normalized with ß-actin. The results are expressed as RQ, mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction (*P < 0.05, **P < 0.005, ***P < 0.0005). G: Protein expression levels of LOX-1, p62, and TRAF6 in MCECs after treatment with either LDb-LDL or LTp-LDL. Protein levels were analyzed by SDS-PAGE followed by Western blot analysis. The results are expressed as mean ± SD. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction. H: Protein levels of CCL-2 and IL-6 in the cell media after treatment with PBS (circles), LDb-LDL (squares), or LTp-LDL (triangles). ELISA was used to determine the protein levels of CCL-2 (in nanograms per milliliter) and IL-6 (in picograms per milliliter) in cell media. Individual measurements are shown as well as mean ± SEM. Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction.

Fig 4: A: The apoB secretion rate is decreased in LTp triple KO mice deficient in PCSK9. Primary hepatocytes were isolated from C57BL/6J, LDb, and LTp mice. Cells were plated onto a 6-well plate coated with mouse type IV collagen. The next day, cells were labeled with 35S-methionine/cysteine for 30 min and chased for 30, 60, 120, 180, and 240 min. Cell media were immunoprecipitated with anti-mouse apoB (left) or anti-albumin (right) antibodies and protein A agarose, followed by SDS-PAGE. The bands of apoB100 (LDb and LTp mice) and apoB48 (C57BL/6J mice) were scanned using Typhoon FLA 7000 (GE) and quantified using Quantity One software (Bio-Rad). The results are expressed as total radioactivity in each band (mean ± SD). Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction. ❖P < 0.05, comparing LDb to LTp mice. The experiments were performed three times with duplicate samples for each time point. The radioactivities of apoB were too low to detect in cell lysates. The radioactivities of apoB100 in C57BL/6 were too low to measure in cell media. A representative gel image is shown. B: Recombinant PCSK9 increased the apoB secretion rate in primary hepatocytes from LDb and LTp mice. Primary hepatocytes were isolated from LDb and LTp mice. Cells were plated onto a 6-well plate coated with mouse type IV collagen. The next day, the cells were incubated with 10 μg of recombinant PCSK9 for 4 h and the media were removed and washed. Next, the cells were labeled with 35S-methionine/cysteine for 30 min and chased for 30, 60, 120, 180, and 240 min. Cell media were immunoprecipitated with anti-mouse apoB (left) or anti-albumin (right) antibodies and protein A agarose, followed by SDS-PAGE. The bands of apoB100 (LDb and LTp mice) were scanned by Typhoon FLA 7000 (GE) and quantified using ImageQuant TL8.1 software (GE). The experiments were performed three times and the results are expressed as total radioactivity of each band (mean ± SD). Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction. ❖P < 0.05, comparing LDb versus LDb incubated with PCSK9 and LTp versus LTp incubated with PCSK9. C: There is no significant difference in hepatic apoB mRNA levels between LDb and LTp mice. Hepatic apoB mRNA levels in LDb (n = 3) and LTp (n = 3) mice were determined using real-time quantitative RT-PCR. The results are presented as RQ (apoB mRNA normalized with 18S-RNA; mean ± SD). Statistical analyses were performed using two-tailed unpaired t-tests with Welch’s correction. The differences are not significant (ns).

Fig 5: A schematic diagram on regulation of PCSK9 on apoB via modulating the autophagy pathway affecting atherogenesis. 1: In the liver, PCSK9 interacts with apoB, which inhibits the normal degradation of apoB via the autophagy pathway involving p62. The excess apoB assembles and secretes as VLDL into the circulation. 2: The regulation of PCSK9 on apoB on assembly and secretion as VLDL intracellularly might activate p-Akt, resulting in suppression of autophagy. On the other hand, this process might induce AMPK and p-AMPK. However, the activation of AMPK and p-AMPK also induces the ULK1 kinase complex and activates Beclin-1 to activate autophagy. In this condition, the regulatory element, Atg14L, in the Beclin-1 complex was not activated. Therefore, the signaling pathway through AMPK did not activate autophagy. Thus, p62 is accumulated in the autophagosome; not degraded, LC3-I is converted to LC3-II inefficiently. Much work is needed to elucidate this complex hypothesis. The role of mTOR in this hypothesis is not confirmed yet. 3: The VLDLs secreted from liver have different compositions as the result of presence of PCSK9. VLDLs are hydrolyzed to LDLs; the compositions of LDL generated from LDb versus LTp mice are different; LTp-LDL has fewer CEs and PLs. 4: LDb-LDL and LTp-LDL induce different activations on ECs and immune cells, leading to inflammatory reaction of macrophages (MQs) and migration/proliferation of smooth muscle cells (SMCs). This is the initiation of atherogenesis.