PROTAC-mediated PCSK9 degradation attenuates atherosclerosis and improves plaque composition via suppression of NF-κB/TNF-α pathway

Abstract

Background

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a critical therapeutic target for managing hyperlipidemia and atherosclerosis. We developed Cadd4, a synthetic proteolysis-targeting chimera (PROTAC) engineered to selectively induce proteasomal degradation of the PCSK9 protein. In this study, we investigate Cadd4’s anti-atherosclerotic properties and concurrently evaluate the feasibility and safety of its long-term therapeutic administration.

Methods

Cadd4-mediated PCSK9 degradation was assessed in mouse monocyte-macrophage leukemia cells(RAW264.7), mouse aortic vascular smooth muscle cells (MOVAS) and human umbilical vein endothelial cells (HUVECs) using immunofluorescence. Its anti-inflammatory effects were examined in lipopolysaccharide (LPS)-stimulated cells via quantitative real‑time PCR (qRT-PCR) and western blot. In vivo efficacy was assessed in apolipoprotein E-deficient (ApoE^−/−) mice maintained on a high-fat diet (HFD). Animals received intraperitoneal injections of Cadd4 (20 mg/kg every two days) or subcutaneous injections of alirocumab (3 mg/kg weekly) for 12 consecutive weeks.

Results

Cadd4 induced dose-dependent PCSK9 degradation in all three cell types tested and significantly attenuated LPS-induced inflammatory responses. Notably, a 2-week intraperitoneal administration of Cadd4 led to a marked reduction in PCSK9 expression in both the liver and aorta of treated mice. In HFD-fed ApoE^−/− mice, 12-week administration of Cadd4 significantly decreased atherosclerotic plaque area, enhanced collagen deposition within plaques and suppressed intra-plaque inflammation. Importantly, compared with alirocumab, Cadd4 demonstrated superior efficacy in suppressing matrix metalloproteinase (MMP) expression and increasing collagen content, effects that are likely mediated via inhibition of the NF-κB/TNF-α signaling pathway. Of note, Cadd4 mediated PCSK9 modulation did not alter plasma lipid profiles in this model. Collectively, these anti-atherosclerotic effects underscore the lipid-independent anti-inflammatory activity and plaque composition-improving capacity of Cadd4.

Conclusions

Cadd4 potently induces PCSK9 degradation in arterial tissues, mitigates atherosclerotic progression and improves plaque composition via lipid-independent inhibition of the NF-κB/TNF-α pathway. These findings underscore the therapeutic promise of Cadd4 as a candidate for managing atherosclerotic cardiovascular disease.

Background

Atherosclerosis remains a leading cause of global cardiovascular morbidity and mortality, driven by a complex interplay of pathophysiological processes [1]. Its pathogenesis is characterized by endothelial activation, followed by macrophage recruitment, foam cell formation and lipid accumulation, ultimately culminating in chronic arterial inflammation [2, 3]. This pathological cascade perpetuates vascular stenosis, plaque progression and destabilization [4], thereby positioning atherosclerosis as the predominant underlying pathology of major adverse cardiovascular events (MACE) [5].

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a critical regulator of cholesterol metabolism, acting through hepatic low-density lipoprotein receptor (LDLR) degradation and subsequent elevation of circulating low-density lipoprotein cholesterol (LDL-C) [3, 6, 7]. Beyond its canonical role in lipid homeostasis, emerging evidence demonstrates that PCSK9 directly promotes inflammatory response by stimulating the secretion of proinflammatory cytokines, including TNF-α and IL-6, from macrophages and smooth muscle cells [8–12]. This cytokine-driven amplification loop perpetuates vascular inflammation and acts synergistically with dyslipidemia to accelerate atherosclerotic plaque formation. Accordingly, targeted PCSK9 blockade confers both lipid-lowering and anti-inflammatory benefits in the management of atherosclerosis [13–16]. Notably, certain experimental models, such as ApoE^−/− mice, enable the dissection of these pathways, as PCSK9 knockout in this setting does not alter circulating lipid profiles [10, 17], thereby offering a unique platform to investigate its lipid-independent anti-inflammatory and atheroprotective effects. This model was therefore employed in the present study to specifically evaluate the non-lipid mechanisms by which Cadd4-mediated PCSK9 degradation attenuates atherosclerosis.

Current pharmacological strategies targeting PCSK9 include monoclonal antibody inhibitors (e.g., alirocumab, evolocumab) and small interfering RNA (siRNA)-based agents such as inclisiran. These interventions achieve substantial LDL-C reductions of 55–60%, with the advantage of markedly extended dosing intervals [18, 19]. Despite their therapeutic efficacy, several limitations continue to constrain their broader clinical adoption. These limitations include high annual costs that substantially exceed those of conventional statins as well as safety concerns associated with siRNA off-target effects, particularly lipid nanoparticle accumulation in extrahepatic tissues [20]. Additional drawbacks involve compensatory feedback-induced elevations in circulating PCSK9 levels following monoclonal antibody therapy [21–25].

The exploration of novel strategies for PCSK9 inhibition or intracellular degradation represents a promising frontier in PCSK9-targeted drug development [26]. Proteolysis-targeting chimera (PROTAC) technology, using bi-functional molecules to recruit E3 ubiquitin ligases for selective ubiquitin-proteasomal degradation of target proteins, has garnered considerable therapeutic interest [25, 27]. PROTACs offer potential advantages over existing modalities, including high target specificity, efficient protein degradation, minimal compensatory feedback and cost-effective production [25, 28]. This technology has advanced to clinical trials for chronic conditions including cancers, neurodegenerative disorders and autoimmune diseases [29–31].

We recently established an integrated computational-experimental framework combining structure-based docking, pharmacophore modeling and synthetic chemistry to develop a rational PROTAC design platform. Through this computer-aided drug design (CADD) pipeline, we engineered four PCSK9 degraders (Cadd1-4), identifying Cadd4 as a first-in-class heterobifunctional PCSK9 degrader [7, 32]. Both in vitro and in vivo tests demonstrated that Cadd4-mediated PCSK9 degradation confers anti-inflammatory effects and improves plaque composition. These findings provide initial validation of Cadd4’s anti-atherosclerotic efficacy, supporting its potential as a promising therapeutic candidate for atherosclerosis.

Methods

Computational design and synthesis of Cadd4

Based on our established CADD platform, we computationally designed and synthesized the peptide-based degrader Cadd4 targeting PCSK9, following the strategy detailed in our previous publication [7]. Briefly, a 10-mer peptide (TSWEEYLDWV) with high predicted affinity for PCSK9 was identified through structure-guided screening. To construct the functional degrader, this peptide was conjugated with a rhodamine fluorophore for visualization and the ALAPYIP ligand for recruiting the VHL E3 ubiquitin ligase complex, using a GSGS linker. The final compound was synthesized by SynPeptide Co. Ltd (Nanjing, China), with purity (>95%) and identity confirmed by HPLC and ESI-MS [7].

Reagents

Lipopolysaccharide (LPS, L2630) and MG-132 (M7449) were purchased from Sigma-Aldrich (MO, USA). Alirocumab (S20190042) was obtained from Sanofi Winthrop Industrie (Le Trait, France). Primary antibodies used in this study included: anti-PCSK9 (A00085-2) and anti-β-actin (BA2305) from Boster Biological Technology (Wuhan, China); anti-TNF-α (sc-52746), anti-p65 (sc-8008) and anti-p-p65 (sc-136548) from Santa Cruz Biotechnology (CA, USA); anti-IL-6 (ab290735) from abcam (Cambridge, UK); anti-IKK-α (84372-4-RR) from Proteintech (Wuhan, China) and anti-p-IKK-α (AF3012) from Affinity Biosciences (Jiangsu, China). Secondary antibodies were obtained from Jackson ImmunoResearch (PA, USA).

Cell lines and cell culture

Mouse monocyte-macrophage leukemia cells (RAW 264.7), mouse aortic smooth muscle cells (MOVAS) and human umbilical vein endothelial cells (HUVEC) (BLUEFBIO, Shanghai, China) were maintained in Dulbecco’s modified Eagle’s medium (DMEM) or endothelial cell medium (ScienCell, CA, USA) supplemented with 10% fetal bovine serum (Gibco, CA, USA) at 37 °C in 5% CO₂ humidified atmosphere.

Animals

Eight-week-old male C57BL/6J mice and ApoE^−/− mice (Ziyuan Laboratory Animal Technology, Hangzhou, China) were housed under specific pathogen-free (SPF) conditions (24 °C, 12-h light/dark cycle) with ad libitum access to food and water. All procedures were approved by the Institutional Animal Care and Use Committee of TOPBIOTECH Shenzhen (Ethical Approval Number: TOP-IACUC-2024-0144). After a 28-day acclimatization period on a high-fat diet (HFD), all mice were randomized into three groups. Cadd4 was given via intraperitoneal injection at 20 mg/kg every 48 h (Additional File: Fig. S1, see also [7, 32]). Alirocumab was administered subcutaneously at a dose of 3 mg/kg once per week, according to the established protocol [33, 34]. Following a 12-week intervention, mice were anesthetized by CO₂ inhalation and euthanized. Serum was collected via cardiac puncture. Tissues were harvested for snap-freezing in liquid nitrogen or fixation in 4% paraformaldehyde.

Enzyme-linked immunosorbent assay (ELISA)

Serum PCSK9 levels were measured using a mouse-specific ELISA kit (JL47156, Jonlnbio, Shanghai, China) according to the manufacturer’s protocols.

Serum biochemical analysis

The serum levels of total cholesterol (TC), triglycerides (TG), LDL-C, alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and uric acid (UA) were quantified using a BS-2000M automated biochemistry analyzer (Mindray, China).

Cell viability assay

Cell viability was measured using the CCK-8 Kit (C0038, Beyotime, Shanghai, China), following the manufacturer’s instruction [35]. Briefly, cells were seeded in 96-well plates at a density of 1 × 10⁴ cells/well and cultured for 24 h. Treatments were then added to cell culture medium for designated durations. After treatment, medium was removed and replaced with 100 μL DMEM containing 10 μL CCK-8 solution per well. The plates were incubated at 37 °C for 2 h, followed by absorbance measurement at 450 nm using a microplate spectrophotometer (Synergy H1, BioTek).

Co-immunoprecipitation

Co-immunoprecipitation assays were performed according to previously described methods [36]. Briefly, to extract proteins, cells were lysed with a lysis buffer (Beyotime, Shanghai, China). The concentration of proteins in the lysates was measured using a BCA protein assay kit. Subsequently, the PCSK9 antibody was added to the lysate and incubated overnight at 4 °C. Following this, the lysate was mixed with protein A/G beads and incubated for 3-5 h. The sediment on the beads was washed three times with the lysis buffer and then boiled for 5 min. After boiling, the samples were centrifuged again, and the resulting supernatant was used for the subsequent western blot analysis.

Immunofluorescence and Immunohistochemistry staining

Immunofluorescence was conducted as previously described [37]. Briefly, paraffin-embedded sections were first deparaffinized and rehydrated. Then, they were subjected to heat-induced epitope retrieval using 10 mM citrate buffer at 95 °C for 15 min. After blocking, the sections were incubated overnight at 4 °C with primary antibodies against PCSK9 (1:100), TNF-α (1:100), IL-6 (1:100), and p-p65 (1:100). After several washes with PBS, fluorescent secondary antibodies specific to the species of the primary antibodies were applied and incubated for 1 h at 37 °C in the dark. Next, nuclei were counterstained with DAPI for 5 min at room temperature. Images were acquired using an FV3000 laser-scanning confocal microscope (Olympus). For immunohistochemical detection, a comparable procedure was followed. After the same antigen retrieval process, sections were incubated overnight at 4 °C with primary antibodies against PCSK9 (1:100), CD68 (1:100), and α-SMA (1:100). Subsequently, HRP-conjugated secondary antibodies were applied, followed by DAB chromogenic development and hematoxylin counterstaining.

Hematoxylin and eosin (H&E), Oil-Red-O and Masson staining

H&E, Oil-Red-O and Masson staining were conducted as previously described [3]. Briefly, during morphometric analysis, aortic root sections were subjected to H&E staining (G1108, Servicebio). For plaque size, morphology and lipid content, intact aorta and aortic root sections were stained with Oil-Red-O (O0625, Sigma-Aldrich). The collagen content in plaques was determined using a Masson staining kit (G1346, Servicebio), following the manufacturer’s protocol, where blue indicates collagen fibers and red indicates muscle fibers. Image analysis was performed using ImagePro Plus v.5.1 (Media Cybernetics).

Western blot

Western blot was conducted as previously described [38, 39]. Briefly, total protein extracts from cells and tissues were separated by SDS-PAGE and transferred to PVDF membranes. After blocking for 1 h at room temperature with 5% skim milk, the membranes were incubated overnight at 4 °C with primary antibodies against PCSK9, p65, p-p65, MMP9, MMP2, β-actin or GAPDH (all diluted 1:1000). Following PBS washes, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Protein bands were subsequently detected using an ECL substrate kit (E412-02, Vazyme, China) on an Amersham Imager 600 system.

Quantitative real-time PCR (qRT-PCR)

Total RNA was isolated from cells and tissues using the RNA Extraction Kit (LS1040, Promega) according to the manufacturer’s protocol. The cDNA synthesis was performed with Evo M-MLV RT Premix. For qRT-PCR analysis, SYBR Green Master Mix (AG11701, Accurate Biology) was utilized on a QuantStudio 5 system (Applied Biosystems). The relative mRNA expression levels were normalized to GAPDH and calculated using the 2^−ΔΔCt method. Primer sequences are listed in Supplemental Table 1.

Statistical analysis

All data were analyzed using GraphPad Prism software version 9.0. Normality was assessed using the Shapiro-Wilk test. Normally distributed data are presented as mean ± SEM and were compared between two groups using unpaired t-tests or among multiple groups using one-way ANOVA. Non-normally distributed data are presented as median (interquartile range) and were compared using Mann-Whitney U tests for two groups or Kruskal-Wallis tests for multiple groups. All statistical tests were two-tailed, and a P value < 0.05 was considered statistically significant.

Results

Cadd4 degrades PCSK9 protein via the cellular ubiquitin-proteasomal system

Considering the widespread expression of PCSK9 in plaque-resident macrophages, smooth muscle cells and endothelial cells [40–42], we assessed Cadd4-mediated PCSK9 degradation in these cells. Rhodamine-labeled Cadd4 was applied to RAW264.7, MOVAS and HUVEC cells at concentrations of 10, 20 or 40 μM. Confocal microscopy observations revealed a concentration- and time-dependent cellular uptake of Cadd4, as demonstrated by intracellular rhodamine fluorescence (Fig. 1A-C). Western blot analysis confirmed a corresponding concentration-dependent reduction in PCSK9 protein levels (Fig. 1D-F). Based on the impact on PCSK9 degradation and cell viability (Additional File: Fig. S2A-C), 20 μM Cadd4 was chosen for further experiments.

Fig. 1.

Cadd4 degrades PCSK9 protein via the cellular ubiquitin-proteasomal system. A RAW 264.7 cells, B MOVAS cells and C HUVEC cells were incubated with rhodamine-labeled Cadd4 at different concentrations (10, 20, 40 μM). After incubation for 0, 2, 4 and 8 h, the localization of Cadd4 in cells was observed by confocal laser microscopy (n = 3). Red, rhodamine-labeled Cadd4; blue, the nucleus. The protein levels of PCSK9 in D RAW 264.7 cells, E MOVAS cells and F HUVEC cells were analyzed by western blot after treatment with different concentrations of Cadd4 (0, 10, 20, 40 μM) for 8 h (n = 3). G RAW 264.7 cells, H MOVAS cells and I HUVEC cells were treated with Cadd4 (20 μM) and proteasome inhibitor MG-132 (10 μM) for 8 h, then the whole cell lysates were immunoprecipitated with PCSK9 antibody and the ubiquitination level of PCSK9 was analyzed by western blot. Western blot analysis were used to evaluate the remedial effect of 10 μM MG-132 co-incubation with Cadd4 on PCSK9 expression in J RAW 264.7 cells, K MOVAS cells and L HUVEC cells (n = 4). Data were shown as mean ± SEM. Scale bar, 5 µm for A, B, C. *P < 0.05; **P < 0.01 and ***P < 0.001 by one-way ANOVA D-F, J-L

The designed mechanism of Cadd4 requires the recruitment of the endogenous von Hippel–Lindau (VHL) E3 ubiquitin ligase complex. Here, we confirmed that VHL is expressed in all three cell types by using western blot analysis (Additional File: Fig. S3A-C). To elucidate the molecular basis of PCSK9 degradation, endogenous PCSK9 was immunoprecipitated and ubiquitin immunoblotting demonstrated that Cadd4 treatment markedly increased the levels of ubiquitin-conjugated PCSK9 compared to controls in all cell types (Fig. 1G-I). To determine whether this ubiquitination is mediated by the proteasome, cells were co-treated with Cadd4 and the proteasome inhibitor MG-132. Both western blot and immunofluorescence indicated that Cadd4-induced PCSK9 degradation was blocked following co-treatment with MG-132 (Fig. 1J-L, Additional File: Fig. S4A), thereby confirming ubiquitin-proteasomal mediated degradation.

Cadd4 suppresses LPS-induced cellular inflammation by inhibiting the NF-κB pathway

Based on the known proinflammatory role of PCSK9 [8], we next investigated whether Cadd4 exerts anti-inflammatory activity. In LPS-stimulated RAW264.7, MOVAS and HUVEC cells, mRNA levels of inflammatory cytokines (TNF-α, IL-6, IL-1β), chemokines (CCL2, CXCL1) and matrix metalloproteinases (Mmp2, Mmp9) were significantly elevated. Co-treatment with Cadd4 significantly reduced the expression of these mediators (Fig. 2A-C, Additional File: Fig. S5A-C). To determine whether this anti-inflammatory effect is specifically attributable to PCSK9 degradation, cells were treated with negative control PROTAC Cadd1 (Additional File: Fig. S6A-C; see also [7]). Quantitative real-time PCR (qRT-PCR) analysis indicated that suppression of pro-inflammatory cytokine mRNA levels occurred exclusively in Cadd4-treated cells, with no such effect was observed in cells exposed to Cadd1 (Additional File: Fig. S6D-F).

Fig. 2.

Cadd4 suppresses LPS-induced cellular inflammation by inhibiting the NF-κB pathway. A RAW 264.7 cells and B MOVAS cells were incubated with 1 μg/mL LPS for 12 h and treated with 20 μM Cadd4. The expressions of pro-inflammatory cytokines TNF-α, IL-6, IL-1β and CCL2 were detected by qRT-PCR, and GAPDH was used as the internal control (n = 3). C HUVEC cells were incubated with 1 μg/mL LPS for 12 h and treated with 20 μM Cadd4. The expressions of pro-inflammatory cytokines TNF-α, IL-6, IL-1β and CXCL1 were detected by qRT-PCR (n = 3). The protein levels of p-p65, p65, p-IKK-α, IKK-α, TNF-α and PCSK9 in D RAW 264.7 cells and E MOVAS cells were analyzed by western blot, and β-actin was used as the internal control (n = 3). Data were shown as mean ± SEM. *P < 0.05; **P < 0.01 and ***P < 0.001 by one-way ANOVA A-E

In view of the central role of the NF-κB pathway in mediating inflammatory responses, we next examined whether Cadd4 modulates this signaling cascade. Western blot analysis confirmed that Cadd4 co-treatment effectively reduced PCSK9 protein levels and phosphorylation of key NF-κB pathway components, including IKK-α and p65 (Fig. 2D-E). This suppression of NF-κB pathway activation was accompanied by a concomitant decrease in TNF-α protein production (Fig. 2D-E). Furthermore, qRT-PCR analysis of key NLRP3 inflammasome components (NLRP3 and Caspase-1) revealed a modest reduction in their mRNA expression following Cadd4 treatment in RAW264.7 and MOVAS (Additional File: Fig. S7A-C). Collectively, these findings suggest that the potent anti-inflammatory efficacy of Cadd4, while potentially involving additional pathways, is primarily attributable to its marked suppression of the NF-κB/TNF-α signaling axis.

Cadd4 reduces PCSK9 levels in murine aortic and hepatic tissues

To assess Cadd4-mediated PCSK9 degradation in vivo, C57BL/6J mice received intraperitoneal injections of either Cadd4 or saline (control) (Fig. 3A). Western blot analysis revealed significantly reduced PCSK9 protein levels in aortic and hepatic tissues from Cadd4-treated mice compared relative to controls (Fig. 3B-C). Immunofluorescence staining analysis corroborated these findings (Fig. 3D-E), confirming the in vivo efficacy of Cadd4 in promoting PCSK9 degradation.

Fig. 3.

Cadd4 reduces PCSK9 levels in murine aortic and hepatic tissues. A Animal treatment diagram: C57BL/6J mice were intraperitoneally injected with either normal saline or Cadd4, once every two days, and after 14 days, mouse vascular and liver tissues were collected. B Western blotting analysis of PCSK9 protein in the aorta of mice (n = 6 mice). C Western blot analysis of PCSK9 protein in the liver of mice (n = 6 mice). D-E The levels of PCSK9 in the aorta and liver of mice were analyzed by immunofluorescence staining (n = 6 mice). Data were shown as mean ± SEM. Scale bar, 100 µm for D and E, *P < 0.05; **P < 0.01; and ***P < 0.001 by unpaired t test B-E

Cadd4 reduces atherosclerotic burden in HFD-fed ApoE^−/− mice

To evaluate the anti-atherosclerotic efficacy of Cadd4, HFD-fed ApoE^−/− mice mice were administered Cadd4, alirocumab, or saline (control) (Fig. 4A). Both treatment regimens were well-tolerated, and the body weight of the treated mice in both groups was not affected compared to the control group (Fig. 4B). Consistent with previous reports [10, 17], neither treatment significantly altered plasma lipid profiles (Fig. 4C-E). Notably, Cadd4 treatment led to a marked reduction in serum PCSK9 levels, whereas alirocumab did not produce significant decrease compared with controls (Fig. 4F). Oil Red O staining revealed a substantial reduction in plaque area within the whole aorta, aortic arch, and aortic root of Cadd4-treated mice (Fig. 4G-I). H&E staining of aortic root sections further demonstrated significantly smaller necrotic cores in both treatment groups (Fig. 4J). Together, these findings indicate that Cadd4 mediates attenuation of plaque progression through a lipid-independent mechanism.

Fig. 4.

Cadd4 reduces atherosclerotic burden in HFD-fed ApoE^−/− mice. A Modeling diagram: ApoE^−/− mice were fed a high-fat diet at 8 weeks of age. After 28 days, the mice were divided into 3 groups and treated with normal saline, Cadd4 and aliciumab for 56 days respectively. B Body weight of Cadd4- and aliciumab-treated and control mice (n = 8 mice). C-F Serum levels of TG, TC, LDL-C (n = 8 mice) and PCSK9 (n = 6 mice) in mice. G-H Oil red O staining images of aortic atherosclerotic plaques and statistical analysis results (n = 3 mice). I Plaque size in aortic root measured by oil red O staining and statistical analysis results (n = 8 mice). J Measurement of necrotic core in aortic root lesions by H&E staining and statistical analysis (n = 8 mice). Data were shown as mean ± SEM. Scale bar, 2 mm for G, 500 µm for I, J, *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA B-F,H-J

Cadd4 improves atherosclerotic plaque composition

To investigate the effect of Cadd4 on the plaque composition, we first examined the levels of PCSK9 protein within atherosclerotic plaques. Immunohistochemical analysis revealed significantly reduced PCSK9 expression in plaques from Cadd4-treated mice compared to both control and alirocumab-treated groups. (Fig. 5A-B). Furthermore, both Cadd4 and alirocumab treatment showed similar reductions in CD68⁺ macrophage infiltration (Fig. 5C-D), whereas no significant differences in α-SMA expression were observed between the two treatment groups (Fig. 5E-F). These results suggest that the blockade or degradation of PCSK9 protein may potentially decrease macrophage accumulation and suppresses intraplaque inflammation.

Fig. 5.

Cadd4 improves atherosclerotic plaque composition. A-F Immunohistochemical images and statistical analysis results of PCSK9, CD68 and α-SMA in aortic root lesions (n = 6 mice). G-H Masson staining and statistical analysis results of collagen in aortic root lesions (n = 8 mice). I-K Western blotting analysis of MMP9 and MMP2 protein in the aorta of mice (n = 6 mice). Data were shown as mean ± SEM. Scale bar, 100 µm for A, C, E, G, *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA B,D,F,H,J-K

Next, We next evaluated collagen content and matrix metalloproteinase (MMP) expression. Masson staining revealed increased collagen deposition in plaques from both treatment groups compared to controls, with Cadd4 demonstrating superior efficacy to alirocumab (Fig. 5G-H). Western blot analysis showed significantly reduced MMP2 expression in Cadd4-treated plaques compared to controls (Fig. 5I-K). In contrast, alirocumab did not significantly alter the expression of either MMP9 or MMP2. Collectively, these results show that Cadd4 treatment is associated with reduced macrophage infiltration, decreased MMP expression, and increased collagen content in plaques. Notably, these findings suggest the plaque-stabilizing potential of Cadd4.

Cadd4 suppresses the NF-κB p65/TNF-α axis

Immunofluorescence analysis of aortic tissues demonstrated significantly reduced levels of TNF-α, IL-6, and phosphorylated p65 (p-p65) in Cadd4-treated mice versus controls (Fig. 6A-F). Western blot analysis corroborated the attenuation of p65 and IKK-α phosphorylation (Fig. 6G-K). These findings indicate that Cadd4-mediated PCSK9 degradation attenuates vascular inflammation by inhibiting the NF-κB pathway, which may contribute to reduced atherosclerotic plaque formation.

Fig. 6.

Cadd4 suppresses the NF-κB p65/TNF-α axis. A-F Analysis of expression of TNF-α, IL-6 and p-p65 in aortic root tissues of mice in normal saline, Cadd4 and alirocumab groups by fluorescence staining (n = 6 mice). G-K The expression levels of p65, p-p65, IKK-ɑ, p-IKK-ɑ in aorta tissue of mice in normal saline, Cadd4 and alirocumab groups were detected by western blot (n = 6 mice). Data were shown as mean ± SEM. Scale bar, 100 µm for A, C, E, *P < 0.05; **P < 0.01; and ***P < 0.001 by one-way ANOVA B,D,F,H-K

Cadd4 exhibits favorable biosafety profiles during long-term administrationt

Evaluation of Cadd4 safety assessment in atherosclerotic mice revealed no significant changes in plasma biomarkers of hepatic function (ALT, AST) or renal function (BUN, UA) following Cadd4 treatment (Fig. 7A-D). Moreover, histological examination of major organs (heart, liver, spleen, lung, kidney) via H&E staining showed no structural abnormalities, inflammatory infiltrates or necrotic change (Fig. 7E). Collectively, these findings confirm Cadd4’s favorable in vivo safety profile, further supporting its therapeutic potential for the clinical management of atherosclerotic disease.

Fig. 7.

Cadd4 exhibits favorable biosafety profiles during long-term administrationt. A-D Biochemical analysis was performed to detect the levels of ALT, AST, BUN and UA in mouse plasma (one-way ANOVA; n = 8 mice) E H&E staining of mouse heart, liver, spleen, lung, and kidney tissue sections. Data were shown as mean ± SEM. Scale bar, 100 µm for E

Discussion

Atherosclerosis is a chronic inflammatory disorder of the arterial intima driven by lipid dysregulation, characterized by impaired lipid homeostasis and aberrant inflammatory signaling [43, 44]. PCSK9 has emerged as a key contributor to atherosclerosis, exerting dual deleterious effects through exacerbation of circulating lipid dysregulation and cholesterol-independent induction of proinflammatory responses in vascular tissues, thereby accelerating plaque formation [45–47]. Consequently, molecular targeting of PCSK9 represents a major therapeutic strategy against atherosclerosis [48].

Current pharmacological agents targeting PCSK9, namely monoclonal antibodies and siRNA-based therapeutics, operate through distinct mechanisms: antibodies antagonize extracellular PCSK9 by blocking its binding to LDLR, whereas siRNA agents suppress hepatic PCSK9 synthesis. Monoclonal antibodies, however, fail to degrade circulating or intracellular PCSK9, leaving intracellular proinflammatory signaling inadequately restrained. Conversely, siRNA-based approaches are constrained by off-target effects, genotoxicity concerns, and prohibitive costs, all of which limit their widespread clinical adoption [49, 50]. In pursuit of novel therapeutic strategies targeting PCSK9, we previously developed a PROTAC-based platform enabling catalytic degradation of PCSK9 via recruitment of E3 ubiquitin ligases and subsequent ubiquitin-proteasomal processing, culminating in the identification of the peptide degrader Cadd4 [7]. While in vivo studies confirmed robust lipid-lowering efficacy [7], the anti-inflammatory and anti-atherosclerotic potential of Cadd4 had not been investigated.

The present study demonstrates that Cadd4 mediates PCSK9 degradation in vascular and hepatic tissues and, importantly, reveals its potent anti-inflammatory and anti-atherosclerotic activity. Notably, in contrast to alirocumab which exclusively neutralizes extracellular PCSK9 [51], Cadd4 promotes significant intracellular PCSK9 degradation, leading to concomitant reductions in both cytosolic and circulating PCSK9 levels [7]. Interestingly, Cadd4 displayed superior plaque-stabilizing efficacy than alirocumab, reflected by reduced MMP2 activity and enhanced collagen deposition within lesions. It should be noted, however, that the differing dosing regimens and routes of administration for Cadd4 and alirocumab preclude a direct, equipotent comparison of target engagement. These regimens and administration routes were selected on the basis of their respective pharmacokinetic profiles and established preclinical efficacy. Future studies incorporating matched dosing schedules will be necessary to enable rigorous comparative efficacy assessments.

Building upon prior evidence documenting the proinflammatory role of PCSK9 and the anti-inflammatory effects of its inhibition, the current study demonstrates that Cadd4 attenuates macrophage infiltration within atherosclerotic plaques while suppressing phosphorylation of p65 and IKK-α and dampening downstream TNF-α signaling in aortic tissue. These findings collectively indicate that Cadd4 is associated with reduced inflammatory cell recruitment, decreased cytokine-driven inflammatory signaling, limited plaque expansion, improved plaque composition and enhanced lesion stabilization (Fig. 8). Together with our previous findings that Cadd4 lowers circulatin lipids in non-ApoE-deficient mice [7], these results support a dual-mechanism model wherein Cadd4 exerts both lipid-modulating and lipid-independent anti-inflammatory effects. Preliminary safety evaluations revealed no overt chronic hepatorenal toxicity or histological abnormalities after treatment for 2 months in vivo, underscoring its favorable therapeutic safety in atherosclerosis management.

Fig. 8.

Cadd4 attenuates atherosclerosis through PCSK9 degradation and inhibition of the NF-κB/TNF-α pathway. The schematic illustrates that Cadd4 exerts a protective effect in atherosclerosis through the NF-κB/TNF-α signaling pathway. The diagram indicates that Cadd4 diminishes the inflammatory response of arteries by modulating the degradation of PCSK9 within cells, thereby delaying the progression of atherosclerosis and stabilizing atherosclerotic plaques. Consequently, Cadd4, which mediates PCSK9 degradation via PROTAC technology, may emerge as a novel therapeutic agent for the treatment of atherosclerosis

Beyond its efficacy and safety, PROTAC technology presents significant translational benefits. Peptide-based degraders such as Cadd4 are synthetically accessible via solid-phase peptide synthesis, offering cost-effectiveness compared to biologics requiring complex mammalian cell production [52]. Importantly, PROTACs operate exclusively at post-translationa level, eliminating genotoxicity risks inherent to gene-editing platforms [53, 54]. Although the present study establishes the the promising efficacy and safety of Cadd4 in preclinical models, several translational challenges must be addressed to advance this peptide-based PROTAC toward clinical application [55]. Developing orally bioavailable small-molecule PROTACs represents an active research direction that could enable oral administration of Cadd4 derivatives, significantly improving patient compliance. Future efforts should focus on optimizing the chemical stability and pharmacokinetic properties of Cadd4 through strategies such as cyclization, incorporation of non-natural amino acids, or other structural modifications. In parallel, advanced formulation approaches (e.g., pegylation, sustained-release carriers) or subcutaneous depot systems could extend dosing intervals and enhance clinical practicality. Overcoming these challenges is essential to advance Cadd4 from proof-of-concept to a clinically viable, patient-friendly therapy for atherosclerotic cardiovascular disease.

Although the present findings provide compelling evidence for the anti-atherosclerotic efficacy of Cadd4, several limitations warrant consideration. The study employed ApoE^−/− mice, in which ApoE, a canonical LDLR ligand, is absent and PCSK9 modulation exerts no lipid-lowering effect, thereby precluding assessment of Cadd4-mediated alterations in circulating lipids, particularly cholesterol. Consequently, this model does not fully recapitulate the pathophysiological complexity of human atherosclerosis [17, 56, 57]. Future investigations should therefore extend to humanized models, such as PCSK9-transgenic mice or non-human primates, to verify cross-species conservation of Cadd4 efficacy. Additionally, the pharmacokinetic profile of Cadd4 was not characterized in the present study;subsequent work should determine its elimination half-life to optimize dosing regimens [58–60]. Furthermore, the current investigation focused primarily on the NF-κB p65/TNF-α inflammatory axis within vascular cells and plaque tissue. Given the reported involvement of PCSK9 in other inflammatory pathways such as the NLRP3/caspase-1 axis [61], we assessed the expression of key NLRP3 inflammasome components. qRT-PCR analysis revealed that Cadd4 treatment induced a modest yet statistically significant decrease in NLRP3 and Caspase-1 mRNA levels (Additional File: Fig. S7A-C). This downregulation, however, was substantially less pronounced than the robust inhibition observed for the NF-κB/TNF-α axis, indicating that the anti‑inflammatory effect of Cadd4 is mediated predominantly through suppression of NF-κB/TNF-α signaling. A more comprehensive dissection of inflammatory signaling networks will be required to fully delineate the mechanisms by which PCSK9 degradation confers atheroprotection [62, 63].

Conclusions

In conclusion, this study establishes PROTAC-mediated PCSK9 degradation via Cadd4 as a promising therapeutic strategy for atherosclerosis. Cadd4 is associated with improved plaque composition and attenuated arterial inflammation, potentially through lipid-independent anti-inflammatory mechanisms. These findings suggest Cadd4 as a viable candidate for atherosclerosis management.

Abbreviations

PROTAC

Proteolysis-targeting chimaera

PCSK9

Protein convertase subtilisin/keto type 9

LPS

Lipopolysaccharide

NF-κB

Nuclear factor kappa-B

LDL-C

Low-density lipoprotein cholesterol

LDLR

Low-density lipoprotein receptor

TC

Total cholesterol

ALT

Alanine aminotransferase

AST

Aspartate aminotransferase

BUN

Blood urea nitrogen

UA

Uric acid

ApoE

Apolipoprotein E

TNF-α

Tumor necrosis factor-alpha

IL-6

Interleukin-6

IL-1β

Interleukin-1 beta

CCL2

Chemokine (C-C motif) ligand 2

CXCL1

Chemokine (C-X-C motif) ligand 1

Author contributions

The authors JY, GF, ZHT, and XW conceived and designed the study. XW, YL, QPZ, SLC and PY performed the experiments and analyzed the data. XW, YL, MCZ and JY contributed to writing and revising the manuscript. JY, GF, ZHT, and QPZ provided funding support, while MCZ, MDX, ML and WMG provided technical support. All authors read and approved the final manuscript.

Funding

The present study was supported by the National Natural Science Foundation of China (82170852, 82471623), Shenzhen Medical Research Fund (A2403066), Guangdong Natural Science Foundation (2025A1515010482), Shenzhen Natural Science Foundation (JCYJ20240813114618024, JCYJ20240813114423031), the Major Science and Technology Project of the Nanshan District Health System (NSZD2023044, NSZD2023041, NSZD2024017), the Outstanding Team Fund of the Nanshan District Health System, Shenzhen City (NSZD2025004), and the Municipal Financial Subsidy for Key Medical Discipline Construction in Nanshan District.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

This study was approved by the Ethics Committee of the Institutional Animal Care and Use Committee of TOPBIOTECH Shenzhen, China (Ethical Approval Number: TOP-IACUC-2024-0144).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Supplementary information

The online version contains supplementary material available at 10.1186/s12916-026-04976-9.