Expression of IMD-CBM peptide induces the autophagic degradation of CAV1 (caveolin 1) to inhibit LDL transcytosis and retard diabetic atherosclerosis

ABSTRACT

Atherosclerosis is attributable to a series of diabetes-related complications. CAV1 (caveolin 1)-mediated low-density lipoprotein (LDL) particle transcytosis across endothelial cells (ECs) is the initial step of atherosclerosis. MAP1LC3/LC3-interacting regions in the intramembrane domain (IMD) of CAV1 were buried in the caveolae and were not accessible for LC3B interaction, protecting CAV1 from autophagic degradation. However, the CSD domain of CAV1, exposed in the cytosol, directly interacted with a CBM domain of LC3B and inhibited autophagy. Therefore, the peptide IMD-CBM was constructed to induce the selective autophagic degradation of CAV1 and suppress LDL transcytosis in diabetic atherosclerosis. EC-specific expression of IMD-CBM was achieved using adenovirus. IMD-CBM directly interacted with CAV1 and LC3B in ECs, leading to the selective autophagic degradation of CAV1, activation of autophagy, and subsequent inhibition of LDL transcytosis. IMD-CBM promoted the autophagic degradation of CAV1 and consequently reduced the area of atherosclerotic plaques in apoe^−/− diabetic atherosclerotic mice. Overall, IMD-CBM expedited the autophagic degradation of CAV1 and inhibited high glucose-induced LDL transcytosis, highlighting its potential as a novel translatable strategy for the management of diabetic atherosclerosis.

Abbreviations: ACTB: actin beta; AKT/protein kinase B: AKT serine/threonine kinase; AMPK: 5’-adenosine monophosphate-activated protein kinase; CAV1: caveolin 1; CBM: CAV1-binding motif; CRP: C-reactive protein; CSD: CAV1-scaffolding domain; GFP: green fluorescent protein; HUVEC: human umbilical vein endothelial cell; EC: endothelial cell; FITC: fluorescein isothiocyanate; IL6: interleukin 6; IL10: interleukin 10; IMD: intramembrane domain; LDL: low-density lipoprotein; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NFKB/NF-κB: nuclear factor kappa B; NFKBIA/IκBα: NFKB inhibitor alpha; NO: nitric oxide; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PIK3C3/VPS34: phosphatidylinositol-3-kinase catalytic subunit type 3; Rapa: rapamycin; SAA: serum amyloid A; SQSTM1/p62: sequestosome 1; STZ: streptozotocin; TEM: transmission electron microscopy; TNF/TNF-α: tumor necrosis factor.

Introduction

Atherosclerotic cardiovascular diseases (ASCVD) are associated with diabetes mellitus, which acts as an important and independent risk factor [1]. Large-scale clinical studies have identified ASCVD as the primary factor contributing to mortality in patients with diabetes [2]. These patients have a 2–4 times higher risk of atherosclerosis compared with patients without diabetes [3,4], with the early onset and rapid progression of atherosclerotic cardiovascular disease being responsible for high mortality rates due to a lack of effective treatment options [5].

At the initial stage of atherogenesis, low-density lipoproteins (LDLs) traverse through endothelial cells and are deposited underneath the endothelium [6,7]. As the diameter of LDLs is too large to cross the endothelial gap, they can only traverse the endothelial barrier via transcytosis [8,9]. Moreover, LDL transcytosis is mediated mainly by caveolin-1 (CAV1) [10].

CAV1, a membrane protein with a molecular mass of 21–22 kDa, is a crucial structural constituent within caveolae [11]. The N- and C-terminal regions of CAV1 are free in the cytoplasm, in the middle of which is a CAV1-scaffolding domain (CSD, 82–101 amino acids) and an intramembrane domain (IMD, 102–134 amino acids) [12]. CAV1 upregulation promotes proinflammatory factor-induced LDL transcytosis, whereas its deficiency inhibits LDL transendothelial transport [13–16].

Macroautophagy/autophagy, a highly conserved catabolic process, facilitates the transportation of misfolded proteins, damaged organelles, and other macromolecules to the lysosome. These components undergo degradation and subsequent recycling within the lysosome via the creation of double-membraned autophagosomes [17–19]. Autophagosome formation requires 2 ubiquitin-like coupling pathways [20]. The first system leads to ATG12–ATG5 ligation followed by the formation of the ATG12–ATG5-ATG16L1 complex, which interacts with the outer membrane of the phagophore [21,22]. The second system encodes mammalian homologs of the yeast Atg8 gene, which leads to the processing of LC3. When autophagy is induced, proLC3 is hydrolyzed and cleaved by ATG4 to produce LC3-I. LC3-I is processed and modified by the ubiquitin-like system proteins ATG7 and ATG3 to generate LC3-II, which is then recruited into the growing phagophore. LC3-II is present on both the exterior and interior surfaces of autophagosomes. The extension of the phagophroe and completion of the autophagosome cannot be separated from LC3-II generation [20].

A concise peptide motif, LC3-interacting region (LIR), appears repeatedly in autophagy-related proteins, and proteins with the LIR motif directly bind to LC3 via the LIR [23,24]. The signal protein is directly associated with the CSD in CAV1 through a marker peptide sequence known as the CAV1-binding motif (CBM) [25,26]. In our previous study, both CSD and IMD were found to contain multiple LIRs, and LC3B was found to contain the CBM domain. LC3B obstructs autophagy by connecting with CSD and induces autophagy by binding with the IMD [10]. Under physiological conditions, most of the CAV1 is in the cell membrane, and the IMD is even concealed in the cell membrane, making it unable to bind to LC3B and trigger autophagic degradation. In the process of mediating LDL transcytosis into ECs, caveolae are protected from autophagic degradation by the LIRs of CAV1 concealed within them. Thus, it is worth investigating whether a selective CAV1 autophagic degradation receptor can be designed to prompt or guide CAV1/caveolae entry into the autophagosome, followed by consequent degradation.

In our study, we synthesized a novel peptide IMD-CBM. CBM was combined with the CSD in CAV1 to block the combination of endogenous CAV1 and LC3B, and IMD was combined with LC3B to activate autophagy. This led to the formation of the LC3B-IMD-CBM-CAV1 complex, which directed CAV1 into autophagosomes and selectively activated the autophagic degradation pathway of CAV1. LDL transcytosis was inhibited through autophagy activation and CAV1 degradation, potentially alleviating atherosclerosis. Therefore, the novel peptide IMD-CBM synthesized in this study shows potential in treating diabetic atherosclerosis.

Results

Synthesis of adenovirus expressing IMD-CBM

The structural schematic diagram of CAV1 and LC3B is shown in Figure 1(A). IMD is a sequential fragment of CAV1, and CBM is a sequential fragment of LC3B. These sequence segments were combined to yield a novel peptide IMD-CBM. The function of IMD-CBM is illustrated schematically in Figure 1(B). Next, an adenovirus was synthesized to express IMD-CBM (Figure 1(C)). Pre-experiments were used to determine the optimal multiplicity of infection (MOI) and intervention time for Ad-IMD-CBM (Fig. S1). Next, the expression of IMD-CBM was determined using western blotting. The control adenovirus carrying green fluorescent protein (GFP) was 27 kDa, and the Ad-IMD-CBM was approximately 36 kDa (Figure 1(D)). In addition, the expression of IMD-CBM was observed using confocal microscopy. IMD-CBM was mainly expressed on the cell membrane, and a slight fluorescent green signal was observed in the cytoplasm (Figure 1(E)). These results indicated that Ad-IMD-CBM could effectively overexpress IMD-CBM in ECs, which predominantly localizes to the cell membrane.

Figure 1.

Synthesis and expression of IMD-CBM. (A) schematic representation of the structure of IMD-CBM. (B) schematic diagram of the mechanism of IMD-CBM. (C) Schematic representation of adenoviral vectors. (D-E) HUVECs were cultured in 25 mmol/L glucose and transfected with Ad-vehicle and Ad-IMD-CBM for 16 h, IMD-CBM expression was determined using western blotting (D) and confocal microscopy (E). scale bar: 10 nm.

Moreover, the influence of Ad-IMD-CBM on the modulation of inflammatory factors associated with atherosclerosis was investigated. The impact of Ad-IMD-CBM on immune-related cytokines (proinflammatory factors TNF/TNF-α [tumor necrosis factor] and IL6 [interleukin 6] and the anti-inflammatory factor IL10 [interleukin 10]) was evaluated using enzyme-linked immunosorbent assays (ELISA). The expression of CRP (C-reactive protein), SAA (serum amyloid A), and the transcription factors related to the NFKB/NF-κB (nuclear factor kappa B) signaling pathway was determined using polymerase chain reaction (PCR). The levels of IL10, TNF, IL6 (Fig. S2A), NFKB, and NFKBIA/IκBα (NFKB inhibitor alpha) (Fig. S2B) showed minimal changes following Ad-IMD-CBM intervention.

IMD-CBM inhibits LDL transcytosis and uptake in human umbilical vein endothelial cells (HUVECs)

Next, the regulatory impact of IMD-CBM on the transendothelial transport of LDL was measured. As shown in Figure 2A, high-glucose conditions induced LDL transcytosis in HUVECs; however, this effect was reversed by IMD-CBM intervention.

Figure 2.

IMD-CBM inhibits high glucose-induced LDL transcytosis and uptake. (A) HUVECs were cultured in 5.5 mmol/L and 25 mmol/L glucose conditions, transfected with Ad-vehicle or Ad-IMD-CBM for 16 h, and treated with FITC-LDL (50 μg/mL) for 3 h to determine LDL transcytosis (n = 5). (B) HUVECs were cultured in 5.5 mmol/L and 25 mmol/L glucose conditions, transfected with Ad-vehicle or Ad-IMD-CBM for 16 h, and treated with DiI-LDL (50 μg/mL) for 12 h. Representative fluorescence images using confocal microscopy showing DiI-LDL uptake in HUVECs. Red fluorescence indicates DiI-LDL puncta, and blue fluorescence indicates nuclei. Scale bars: 100 μm. (C) quantification of DiI-LDL uptake by HUVECs (n = 20). **p < 0.01 versus 5.5 mmol/L glucose + Ad-vehicle, ^##p < 0.01 versus 25 mmol/L glucose + Ad-vehicle.

LDL uptake by ECs plays a crucial role in the process of LDL transcytosis and is a pivotal factor contributing to the development of atherosclerosis [27]. Therefore, the determination of intracellular LDL levels can also be used to evaluate LDL-induced atherosclerosis. After HUVECs were treated with DiI-LDL, the intensity of red fluorescence emitted by cells was quantified using confocal microscopy. High-glucose conditions could significantly increase the red fluorescence intensity in cells. However, the red fluorescence reduced significantly after treatment with IMD-CBM (Figure 2B-C). These results suggested that IMD-CBM reversed the increase in LDL transcytosis in HUVECs resulting from high glucose conditions.

IMD-CBM interacts with CAV1 and LC3B

In our previous study, we demonstrated that CAV1 could bind to the CBM domain in LC3B and that the IMD in CAV1 could bind directly to LC3B to promote autophagy [10]. Therefore, we next verified whether IMD-CBM could bind directly to CAV1 and LC3B to exert its biological functions. Findings from co-immunoprecipitation experiments confirmed that IMD-CBM interacted with both CAV1 and LC3B (Figure 3(A–C)). Confocal microscopy showed that IMD-CBM was mainly expressed in the cell membrane and was co-localized with both CAV1 and LC3B (Figure 3(D–E)). These findings provided further evidence of the interaction of IMD-CBM with both CAV1 and LC3B.

Figure 3.

IMD-CBM directly binds to CAV1 and LC3B. HUVECs were cultured in 25 mmol/L glucose conditions and transfected with Ad-vehicle or Ad-IMD-CBM for 16 h. (A) immunoblots showing GFP and CAV1 levels in HUVECs precipitated with anti-CAV1 or IgG (n = 5). (B) immunoblots showing GFP and LC3B levels in HUVECs precipitated with anti-LC3B or IgG (n = 5). (C) immunoblots showing GFP, CAV1, and LC3B levels in HUVECs precipitated with anti-GFP or IgG (n = 5). (D) immunofluorescence staining showing the localization of GFP and CAV1 in HUVECs. Green fluorescence indicates GFP, red fluorescence indicates CAV1 labeled with Cy3, and blue DAPI fluorescence denotes nuclei. (E) immunofluorescence staining showing the localization of GFP and LC3B in HUVECs. Green fluorescence indicates GFP, red fluorescence indicates LC3B labeled with Cy3, and blue DAPI fluorescence denotes nuclei. Scale bar: 10 μm.

IMD-CBM stimulates the autophagic degradation of CAV1

Based on our findings suggesting that CBM in the IMD-CBM binds to CAV1 and that IMD binds to LC3B to form a complex, we hypothesized that this complex would be transported to lysosomes through the action of LC3B for the autophagic degradation of CAV1. Therefore, CAV1 levels were determined using western blotting after transfecting HUVECs with Ad-IMD-CBM. IMD-CBM significantly reduced CAV1 expression (Figure 4(A,B)). Next, cycloheximide (CHX) was used to determine the half-life of CAV1 after pretreatment with IMD-CBM. A reduction in the half-life of CAV1 from 8.43 h to 4.77 h was noted upon IMD-CBM overexpression (Figure 4(C,D)). Additionally, immunoelectron microscopy (IEM) images suggested that IMD-CBM promoted CAV1 aggregation in autolysosomes, with a more pronounced effect in a high-glucose environment (Figure 4(E,F)). Last, the promotion of GFP-CAV1 accumulation in acidic LysoTracker-positive organelles due to IMD-CBM was observed (Figure 4(G,H)). These findings demonstrated that IMD-CBM impaired the stability of CAV1 and subsequently stimulated autophagic degradation.

Figure 4.

IMD-CBM stimulates the autophagic degradation of CAV1. (A) western blotting showing CAV1 levels in HUVECs after transfection with Ad-vehicle or Ad-IMD-CBM. (B) Bar graph showing the expression of CAV1 relative to ACTB (n = 5). **p < 0.01 versus Ad-vehicle. (C) HUVECs were treated with CHX after transfection with Ad-vehicle or Ad-IMD-CBM. Cell lysates were collected for western blotting at the indicated time points (n = 5). (D) CAV1 degradation curve (n = 5). (E) HUVECs were transfected with Ad-vehicle or Ad-IMD-CBM, then transfected with CAV1-flag plasmid, and analyzed using immuno-electron microscopy with primary antibodies against the flag and 5-nm colloidal gold-conjugated secondary antibody. Red arrows indicate the 5-nm colloidal gold particles in autolysosomes and/or autophagosomes. Green arrows indicate the 5-nm colloidal gold particles in cytoplasm. Scale bar: 500 nm. (F) Statistical graph for (E) (n = 5). *p < 0.05, **p < 0.01 versus LG + Ad-vehicle, ^##p < 0.01 versus HG + Ad-vehicle in CAV1 in autolysosomes; ^Tp < 0.05 versus LG + Ad-vehicle, ^φφp < 0.01 versus HG + Ad-vehicle in CAV1 in cytoplasm. (G) HUVECs were transfected with GFP-CAV1 plasmids and then transfected with Ad-vehicle or Ad-IMD-CBM, followed by treatment with LysoTracker (100 nM) for1 h. Fluorescence images are shown. Scale bar: 10 μm. (H) Statistical graph for (G) (n = 50). **p < 0.01 versus Ad-vehicle in GFP-CAV1, ^##p < 0.01 versus Ad-vehicle in CAV1 in autolysosomes.

IMD-CBM promotes autophagy through CAV1 degradation

CAV1 reduction has been demonstrated to promote autophagy in endothelial cells [28]. Therefore, the effect of IMD-CBM on autophagy in HUVECs was investigated. Autophagy can be identified based on the formation of autophagosomes that engulf cytoplasmic structures and fuse with lysosomes to degrade them [29]. First, the effect of IMD-CBM on autophagosomes and/or autolysosomes was determined using transmission electron microscopy (TEM). IMD-CBM was found to significantly increase the number of autophagosomes and/or autolysosomes in cells and significantly reduce the number of caveolae on the cell membrane in high-glucose conditions (Figure 5(A,B)). Next, the expression of the autophagic receptor and substrate SQSTM1/p62 and autophagic marker LC3B was determined. IMD-CBM decreased SQSTM1/p62 levels and increased LC3B-II levels, indicating the activation of autophagy (Figure 5(C,D)). Then, the pivotal signaling pathways implicated in the process of autophagy were studied. In addition, the phosphoinositide 3-kinase (PI3K)-AKT/protein kinase B-MTOR (mechanistic target of rapamycin kinase), 5’-adenosine monophosphate-activated protein kinase (AMPK)-MTOR-phosphatidylinositol 3-kinase catalytic subunit type 3 (PIK3C3), and NFKB pathways were also studied. The findings from western blotting demonstrated that IMD-CBM treatment decreased the phosphorylation of AKT and MTOR and also increased the phosphorylation of PRKAA/AMPK and the expression of PI3CK3. Consistent with the results from PCR, IMD-CBM had no significant effect on the NFKB pathway (Fig. S2C-D). These findings indicated that IMD-CBM may regulate autophagy via the AMPK-MTOR-PIK3C3 and PI3K-AKT-MTOR signaling pathways.

Figure 5.

IMD-CBM promotes autophagy. (A) transmission electron micrographs showing autophagosomes and/or autolysosomes in HUVECs. Arrows: autophagosomes and/or autolysosomes; asterisks: caveolae. Scale bar: 1 μm. (B) Statistical graph for (A) (n = 5). *p < 0.05 versus LG + Ad-vehicle, ^##p < 0.01 versus HG + Ad-vehicle in autophagosomes/autolysosomes; ^TTp < 0.01 versus LG + Ad-vehicle, ^φφp < 0.01 versus HG + Ad-vehicle in caveolae. (C) immunoblots showing the expression of SQSTM1/p62 and LC3B in HUVECs transfected with Ad-vehicle or Ad-IMD-CBM. (D) relative expression of the protein normalized to ACTB/β-actin (n = 4). **p < 0.01 versus Ad-vehicle. (E) immunoblots showing the expression of CAV1, SQSTM1/p62, and LC3B in HUVECs transfected with Ad-vehicle or Ad-IMD-CBM in the presence or absence of bafilomycin A1 (100 nM). (F) relative expression of the protein normalized to ACTB. *p < 0.05, **p < 0.01 versus Ad-vehicle; ^#p < 0.05, ^##p < 0.01 versus Ad-IMD-CBM (n = 4–6). (G) Representative confocal fluorescence images showing GFP-LC3 puncta in HUVECs co-transfected with GFP-LC3B and Ad-vehicle or Ad-IMD-CBM in the presence or absence of bafilomycin A₁ (100 nM). Scale bar: 10 μm. (H) Statistical plot showing the number of green fluorescent dots in each cell (n = 5). **p < 0.01 versus Ad-vehicle; ^##p < 0.01 versus Ad-IMD-CBM. (I) Representative confocal fluorescence images showing LC3B puncta in HUVECs transduced with Ad-GFP-RFP-LC3 and transfected with Ad-vehicle or Ad-IMD-CBM, and incubated with rapamycin (5 nM) or without rapamycin. Scale bar: 10 μm. (J) number of red or yellow LC3B puncta in merged images (n = 47–50). **p < 0.01 versus Ad-vehicle in GFP⁺ RFP⁺ (autophagosomes); ^##p < 0.01 versus Ad-vehicle in GFP⁻ RFP⁺ (autolysosomes).

Previous results suggested that the LC3B-IMD-CBM-CAV1 complex is guided into autolysosomes for degradation. Therefore, we examined CAV1 expression and autophagic flux following invention with the lysosomal inhibitor bafilomycin A₁. As shown in Figure 5(E,F), bafilomycin A₁ treatment upregulated the levels of CAV1, SQSTM1/p62, and LC3B-II, and this upregulation was enhanced in IMD-CBM-treated cells. Furthermore, bafilomycin A₁ treatment resulted in an increased number of GFP-LC3 puncta after IMD-CBM overexpression (Figure 5(G,H)). Last, a chimeric GFP-RFP-LC3B construct was employed to examine the role of IMD-CBM in autophagic flux. The fluorescent protein GFP in this construct is pH sensitive and gets quenched in the acidic milieu of lysosomes, whereas the RFP of the construct is unaffected. Therefore, only the fluorescence signal of RFP was observed after the fusion of autophagosomes and lysosomes. IMD-CBM treatment markedly increased the quantity of yellow and red puncta in cells similar to that following rapamycin treatment (Figure 5(I,J)). These findings illustrated that IMD-CBM promoted autophagy via the degradation of CAV1.

IMD-CBM ameliorates atherosclerosis in diabetic apoe^−/− mice through the autophagic degradation of CAV1

To further verify the results from in vitro experiments, a diabetic atherosclerosis model was established using apoe^−/− mice, and the effect of IMD-CBM on atherosclerosis was evaluated by injecting the adenovirus (Ad-vehicle or Ad-IMD-CBM) targeting endothelial cells into the tail vein. Figure 6A showed the schematic diagram of animal experiments. Initially, the efficacy of IMD-CBM in alleviating atherosclerosis in mice was tested. Results from the in situ and en-face analysis of atherosclerotic plaques demonstrated that diabetic mice exhibited greater aortic plaque deposition than nondiabetic mice, as evidenced by the Oil Red O staining of the aorta. Furthermore, the aortic plaques of diabetic mice reduced significantly after IMD-CBM intervention (Figure 6(D,E,H)). Similarly, IMD-CBM significantly reduced the plaque area of the aortic root in diabetic mice, as observed based on Oil Red O staining of the aortic root (Figure 6(F,G)).

Figure 6.

IMD-CBM ameliorates atherosclerosis in diabetic apoe^−/− mice. (A) schematic diagram of animal experiments. (B) Representative transmission electron micrographs showing aorta sections of mice. Arrows, autophagosomes or autolysosomes; asterisks, caveolae. Scale bar: 5 μm. (C) Statistical graph for (B) (n = 8). **p < 0.01 versus vehicle + Ad-vehicle, ^##p < 0.01 versus STZ + Ad-vehicle. (D) Representative images of Oil red O-stained aortas (en face) and (E) the percentage of positive regions in each group (n = 8). *p < 0.05, **p < 0.01 versus vehicle + Ad-vehicle; ^#p < 0.05, ^##p < 0.01 versus STZ + Ad-vehicle. (F) Representative images of Oil-red O-stained aortic root sections and (G) the lesion area in each group (n = 8). Scale bars: 100 μm. *p < 0.05, **p < 0.01 versus vehicle + Ad-vehicle; ^#p < 0.05, ^##p < 0.01 versus STZ + Ad-vehicle. (H) Representative in situ aortic arch images of atherosclerotic plaques (arrows) in each group. (I) confocal microscopy images of IMD-CBM (green) with CAV1 (red) in aortic endothelial cells. Scale bar: 100 μm. (J) confocal microscopy images of IMD-CBM (green) with LC3B (red) in aortic endothelial cells. Scale bar: 100 μm. (K) immunohistochemical staining of mice aorta showing the expression of CAV1, SQSTM1/p62, and LC3B. Scale bar: 50 μm. (L) Statistical graph for (K) (n = 8). *p < 0.05, **p < 0.01 versus vehicle + Ad-vehicle, ^#p < 0.01, ^##p < 0.01 versus STZ + Ad-vehicle.

Immunofluorescence studies were conducted by determining the in vivo expression of IMD-CBM and co-localization with CAV1 and LC3B. IMD-CBM was highly expressed in endothelial cells of the aorta (green signal) (Figure 6(I,J)). The red fluorescence, representing CAV1 (Figure 6(I)) or LC3B (Figure 6(J)), colocalized with green fluorescence in aortic endothelial cells, indicating that IMD-CBM binds directly to CAV1 and LC3B. Results from immunohistochemical analysis suggested that IMD-CBM promoted LC3B expression and inhibited the expression of CAV1 and SQSTM1/p62 in the aortas of diabetic mice (Figure 6(K,L)).

TEM images showed that the aorta of diabetic mice had more caveolae and fewer autophagic vacuoles compared with that of nondiabetic mice. Following IMD-CBM intervention, there was a marked increase in autophagic vacuoles and a significant reduction in the number of caveolae, indicating that IMD-CBM promoted the autophagic degradation of CAV1 (Figure 6(B,C)). Last, a reduction in blood glucose levels was noted in diabetic apoe^-/- mice after IMD-CBM intervention (Fig. S4J). In summary, IMD-CBM ameliorated atherosclerosis in diabetic apoe^-/- mice via the autophagic degradation of CAV1.

Discussion

In this study, a novel peptide, IMD-CBM, was synthesized. IMD-CBM promoted the autophagic degradation of CAV1 and increased autophagic flux by binding to CAV1 and LC3B. The outcome was a reduction in LDL transcytosis, consequently mitigating the onset and development of atherosclerosis.

Dyslipidemia is the pathological basis of atherosclerosis [30]. Subendothelial deposition of LDL particles through transcytosis in arterial endothelial cells is the first step toward atherosclerosis [31,32]. Studies have demonstrated a correlation between autophagy and intracellular lipid homeostasis [33,34]. Autophagy is a cellular catabolism for self-protection and it plays a pivotal role in suppressing inflammatory reactions while concurrently facilitating lipid transportation and cholesterol efflux [35]. CAV1 is an important membranous protein located inside caveolae on the cellular membrane and assumes a crucial function in LDL transport across endothelial cells [11,36,37].

CAV1 is a membrane protein that contains CSD and IMD [12]. We previously reported that CBM, a marker peptide sequence of signal proteins directly related to CSD in CAV1 [25,26], is present in LC3B [10]. In addition, most proteins bind to LC3B through the LIR [38], which is present in both CSD and IMD. Moreover, we previously reported that CSD within CAV1 interacts with LC3B to impede the progression of autophagy, whereas IMD within CAV1 interacts with LC3B to stimulate the progression of autophagy [10]. However, CSD interacts with LC3B through CBM, whereas IMD interacts with LC3B through motives other than CBM. The importance of the protein CAV1 in modulating atherosclerosis has been demonstrated across various human, animal, and cellular studies. The lack of CAV1 enhances the merging of autophagosomes with lysosomes, augments autophagic flow, and fosters autophagy, thereby alleviating atherosclerosis [28,37,39–44]. Based on the above studies, a composite peptide was designed to alleviate atherosclerosis by targeting CAV1. This peptide IMD-CBM was synthesized by connecting IMD within CAV1 and CBM within LC3B. The binding of CBM to CSD in CAV1 inhibits the binding of endogenous LC3B and CAV1. IMD binds to LC3B to activate autophagy and guides CAV1 to autophagosomes, selectively activating the autophagic degradation pathway of CAV1 and ultimately delaying the onset and development of atherosclerosis (Figure 7).

Figure 7.

Mechanism of IMD-CBM in the inhibition of LDL transcytosis. (A) in normal glucose conditions, a portion of CAV1 binds to LC3B and undergoes autophagic degradation, whereas the portion creates caveolae that facilitate LDL transcytosis. (B) under high glucose conditions, the binding of CAV1 to LC3B is hindered, leading to a decrease in autophagic degradation and an increase in caveolae formation, ultimately leading to an increase in LDL transcytosis. (C) IMD-CBM intervention enables CAV1 to bind to both LC3B and CAV1. This facilitates the autophagic degradation of CAV1 while reducing the formation of caveolae and decreasing LDL transcytosis.

Peptide synthesis is mainly divided into 2 pathways, namely, chemical synthesis and biosynthesis [45]. Chemical synthesis is challenging as IMD and CBM sequences contain several hydrophobic amino groups; therefore, this unique biosynthesis method was eschewed in this study. Enzymatic hydrolysis and enzymatic catalysis were not applicable as we had spliced peptides from 2 different proteins. Ultimately, we chose genetic engineering and used a virus vector to express the target polypeptide.

Currently, the mainstream viral vector systems include adenovirus (Ad), adeno-associated virus (AAV), lentivirus (LV), and retrovirus (RV) [46]. Both LV and RV belong to the retrovirus family, which can integrate the genome carried by a virus into the host genome so that the host can stably express foreign genes for prolonged periods [47]. Retroviruses are often used in animal experiments; however, to date, no studies have reported their direct use in humans. AAV has low immunogenicity, high safety, and stable expression in the host for 6 months after one transfection, rendering it highly suitable for animal experiments [48]. However, it is routinely not used in cellular experiments because of its low expression and the process being time-consuming. Adenovirus has a high titer, large vector capacity, is not integrated into the chromosome, shows no insertion mutagenicity, and exhibits high expression. It is also suitable for cellular and animal experiments [49]. In addition, adenovirus has been used as a novel corona vaccine vector in humans, further demonstrating its safety [50]. Therefore, the adenovirus was chosen in the current study to express the IMD-CBM composite peptide in both cells and animals.

In our previous study, we reported that IMD could combine with LC3B and that CBM in LC3B could combine with CAV1 for subsequent biological functions [10]. In this study, we spliced IMD and CBM to form the novel IMD-CBM peptide, which could directly combine with CAV1 and LC3B simultaneously, laying the foundation for further functional experiments.

Previous studies have discovered that CAV1 knockdown in endothelial cells promotes autophagic flux and inhibits LDL transcytosis, thereby improving atherosclerosis processes, in which CSD in CAV1 plays a crucial role [10,28,51]. The combinations of CBM in LC3B and CSD activated the autophagy pathway, whereas the combinations of IMD in CAV1 and LC3B had the opposite effect, inhibiting autophagy. IMD is an intramembrane fragment wherein the buried LIR cannot bind to LC3B to fulfill its function; thus, CAV1 functions as a CSD to activate autophagy. Therefore, following IMD-CBM transfection, exogenous IMD expression increased significantly and was completely bound to LC3B for autophagy activation. Furthermore, the combination of CBM and CSD in endogenous CAV1 blocked the combination of endogenous CAV1 and LC3B; however, it formed a CAV1-CBM-IMD-LC3B complex that was capable of transporting CAV1 to lysosomes for selective degradation through LC3B, further activating the selectively autophagy of CAV1. Furthermore, IMD-CBM intervention could increase PRKAA/AMPK phosphorylation, inhibit MTOR activity, upregulate PIK3C3, and inhibit AKT phosphorylation. Previous studies have demonstrated that elevated glucose levels can impede the autophagic degradation of CAV1 by inhibiting the AMPK-MTOR-PIK3C3 pathway. Conversely, CAV1 knockdown can facilitate the activation of the AMPK pathway [10]. Consequently, the regulation of AMPK-MTOR-PIK3C3 by IMD-CBM is potentially mediated by the promotion of CAV1 degradation. Furthermore, activation of the AMPK pathway facilitates the autophagic degradation of CAV1, establishing a positive feedback loop.

In addition to disorders in lipid metabolism, another key factor in the development of atherosclerosis in diabetes is hyperglycemia-induced endothelial cell dysfunction [52]. CAV1 expression is elevated in the hyperglycemic state and affects nitric oxide (NO) production in endothelial cells by interacting with NOS3/eNOS (nitric oxide synthase 3) [53,54]. Conversely, high glucose levels increase oxidative stress and result in an imbalance in NO and reactive oxygen species (ROS) levels [55], further promoting the development of diabetic vascular complications. This study demonstrated that IMD-CBM augmented NO release from apoe^-/- mice endothelial cells (Fig. S3A-C) and HUVECs (Fig. S3D-G) in a high-glucose environment without a notable impact on ROS levels. This outcome may be attributed to the observation that IMD-CBM diminished CAV1 expression in endothelial cells, subsequently enhancing NO production and thereby contributing to the alleviation of endothelial injury.

To further assess the immunogenicity and safety of Ad-IMD-CBM, we quantified plasma levels of the cytokines TNF, IL6, and IL10, as well as the inflammatory markers CRP and SAA in apoe^-/- mice. Results demonstrated that Ad-IMD-CBM transfection caused no significant changes in the levels of these parameters, indicating an absence of systemic immune activation and supporting the safety of Ad-IMD-CBM (Fig. S4A-E). Next, findings from in vivo studies indicated that diabetic mice with atherosclerosis demonstrated lower triglyceride (TG) and total cholesterol (TC) levels following IMD-CBM intervention (Fig. S4F-G). Previous studies have demonstrated that hydrophobic amino acids (residues 101–134) in CAV1, known as IMD, are necessary for lipid droplet targeting [56]. The primary constituents of lipid droplets are triglycerides and sterol esters, in addition to other endogenous neutral lipids, including free cholesterol, which are located in the nuclei of the droplets [57]. Thus, the decrease in TG and TC levels in diabetic mice with atherosclerosis is possibly due to the autophagy of lipid droplets, which is caused by IMD binding to lipid droplets after IMD-CBM transfection.

β-cell dysfunction and insulin resistance are two fundamental pathophysiological mechanisms in type 2 diabetes [58,59], and also play a crucial role in diabetic vascular complications. CAV1 depletion is known to reduce β-cell apoptosis and increase insulin secretion [60], and cav1^−/− diabetic mice exhibit a hyperglycemic, hyperinsulinemic phenotype [61]. Nevertheless, we demonstrated a reduction in blood glucose levels following IMD-CBM intervention. On the one hand, IMD-CBM may indirectly enhance β-cell function by preserving the integrity of the vascular endothelium within pancreatic islets. Promoting the repair of islet vascular endothelial cells helps improve local microcirculation and nutrient delivery to β cells. This, in turn, can partially restore their insulin-secreting capacity and contribute to a mild hypoglycemic effect. On the other hand, IMD-CBM transfection leads to its binding to lipid droplets to cause lipid droplet autophagy. The reduction in lipid droplets enhanced insulin sensitivity and signaling. Additionally, IMD-CBM-induced CAV1 degradation promoted insulin secretion, which led to a tendency of a decrease in blood glucose levels. Lowering blood glucose levels may contribute to the amelioration of atherosclerosis; however, the extent and duration of glycemic reduction achieved in mice in this study was inadequate to produce a significant improvement in atherosclerotic lesions [62–66]. Therefore, the hypoglycemic effect of IMD-CBM is more likely to play a complementary role in attenuating atherosclerosis.

Our study has some limitations. First, the large number of hydrophobic amino groups in IMD-CBM means that the direct synthesis of small molecule peptides is not possible. Thus, viral transfection is required, which increases the complexity of treatment. Second, the mechanism of IMD-CBM in regulating blood glucose and lipid levels requires further experimental exploration. Last, it is unclear whether IMD-CBM affects plaque composition. Therefore, further studies are warranted to elucidate the underlying mechanisms.

In conclusion, the synthesis of an IMD-CBM composite peptide has been presented in this study. IMD was combined with LC3B to activate autophagy and CBM was combined with CSD in CAV1 to block the combination of CSD and LC3B, thereby guiding CAV1 into autophagosomes and selectively activating the autophagic degradation pathway. Through autophagy activation and CAV1 degradation, the transcytosis of LDL was inhibited, potentially alleviating atherosclerosis. Our findings offer a theoretical basis for understanding the mechanisms of diabetes-induced atherosclerosis and highlight the potential of the composite peptide IMD-CBM as a novel prevention and treatment strategy.

Materials and methods

Cell culture

Human umbilical vein endothelial cells (HUVECs) were bought from Cell Biologics (H-6207). Complete medium was configured by adding 10% fetal bovine serum (Yeasen, 40130ES76), 100 U/mL penicillin and 100 mg/mL streptomycin to Dulbecco’s Modified Eagle Medium (Servicebio, G4523). HUVECs were cultured with the configured complete medium at 37°C in an atmosphere of 5% CO₂. Cells were digested with trypsin (Servicebio, G4005) when the cell density reached 90%, followed by seeding in fresh media for subsequent experiments.

Adenovirus construction

The synthesis of adenovirus expressing IMD-CBM was commissioned to Yunzhou Biological Company (Guangzhou, China). The crucial synthesis steps were as follows: adenovirus type 5 (Ad5) was used as a vector. The adenovirus packaging system included a transfer plasmid containing the adenovirus genome sequence with the E1/E3 gene deleted and a packaging cell line expressing the E1 gene. The transfer plasmid carrying the gene of interest was linearized using the endonuclease PacI. The linearized plasmid DNA was then transfected into HEK293A cells (Yunzhou Biological Company), and the expression product of the E1 gene activated the expression of the adenovirus gene on the transfer plasmid (deletion of the E3 gene did not affect the packaging of the adenovirus), resulting in crude recombinant adenovirus particles. The crude adenovirus was again transduced into packaging cells for amplification and then purified and concentrated using cesium chloride (CSCL) density-gradient centrifugation. Viral titers were > 10¹² PFU/mL. The schematic representation of adenoviral vectors is presented in Figure 1(C).

Western blotting analysis

Western blotting was performed following a previously published method [67]. Briefly, the proteins were obtained from specific cells and incubated separately with the following primary antibodies: anti-LC3B (Cell Signaling Technology, 3868), anti-SQSTM1/p62 (Cell Signaling Technology, 39,749), anti-CAV1/caveolin 1 (Cell Signaling Technology, 3267), anti-PRKAA/AMPK (Cell Signaling Technology, 5831), anti-p-PRKAA/AMPK (Cell Signaling Technology, 50,081), anti-MTOR (Cell Signaling Technology, 2972), anti-p-MTOR (Cell Signaling Technology, 2971), anti-PIK3C3/VPS34 (Sigma-Aldrich, V9764), anti-AKT (Cell Signaling Technology, 9272), anti-p-AKT (Cell Signaling Technology, 9271), anti-NFKB (Abmart, T55034), anti-p-NFKB (Abmart, PC0982), anti-NFKBIA/IκBα (Abmart, PC1287), anti-p-NFKBIA/IκBα (Abmart, TC60711), anti-TUBA/α-tubulin (Proteintech, 11,224–1-AP), anti-GAPDH (Proteintech, 10,494–1-AP), anti-ACTB/β-actin (Cell Signaling Technology, 4970) and anti-GFP (Proteintech, 66,002–1-Ig). Subsequently, the secondary antibodies, goat anti-rabbit IgG (Proteintech, SA00001-2) and goat anti-mouse IgG (Proteintech, SA00001-1), were used.

Determination of NO content

Intracellular NO content was determined using DAF-FM DA (NO fluorescent probe; Beyotime, S0019) and confocal microscopy. Briefly, HUVECs were cultured in confocal dishes for 24 h. Next, the cells were transfected with Ad-vehicle and Ad-IMD-CBM for 24 h, respectively. After rinsing 3 times with phosphate-buffered saline (PBS; Servicebio, G4202), the cells were incubated with DAF-FM DA solution (1:1000 dilution) for 30 min, washed 3 times with PBS and fixed in 4% formaldehyde for 30 min. The cells were observed and photographed using confocal microscopy.

Determination of ROS levels

Intracellular ROS levels were determined using DCFH-DA (ROS fluorescent probe; Beyotime, S0033) and confocal microscopy. Briefly, HUVECs were cultured in confocal dishes for 24 h and transfected with Ad-vehicle and Ad-IMD-CBM for 24 h, respectively. After rinsing 3 times with PBS, the cells were incubated with DCFH-DA solution (1:1000 dilution) for 20 min. Next, the cells were washed 3 times with PBS and fixed in 4% formaldehyde for 30 min. The cells were observed and photographed using confocal microscopy.

qPCR

Total RNA was extracted from treated HUVECs using TRIzol (Invitrogen, 15596026CN), and reverse transcription was performed using PrimeScript RT Master Mix (TaKaRa, RR036A). Next, real-time quantitative PCR was conducted using an TB Green® Premix Ex Taq kit (TaKaRa, RR420B), with GAPDH serving as an internal reference. The primer sequences were as follows:

CRP forward: 5’-AGTCACAGTAGCTCCAGTACACATTTG-3’

CRP reverse: 5’-TATCCCTTCTTCAGACTCTTCCTCACC-3’

SAA forward: 5’-GGGACATGTGGAGAGCCTACTC-3’

SAA reverse: 5’-TTCCCCCGAGCATGGAA-3’

NFKB forward: 5’-CCCACGAGCTTGTAGGAAAGG-3’

NFKB reverse: 5’-GGATTCCCAGGTTCTGGAAAC-3’

NFKBIA forward: 5’-CTGCACTTGGCCATCATC-3’

NFKBIA reverse: 5’-GAGTCTGCTGCAGGTTGTTC-3’

GAPDH forward: 5’-GGAAGCTTGTCATCAATGGAAATC-3’

GAPDH reverse: 5’-TGATGACCCTTTTGGCTCCC-3’

Plasmid transfection

GFP-CAV1 and GFP-LC3 plasmids were synthesized by GenScript Biotechnology (Nanjing, China). Cells were transfected with Effectene Transfection Reagent (Qiagen, 301,425), and the plasmids were mixed proportionally according to the manufacturer’s instructions.

Co-immunoprecipitation assay

HUVECs were treated with immunoprecipitation lysis buffer (Beyotime, P0031) for 20 min, following which, the lysates were collected and centrifuged at 14,000 g for 5 min. The supernatant was mixed with protein G/A (Beyotime, P2012) for 15 min and centrifuged again. Next, primary antibodies (anti-GFP, anti-CAV1, anti-LC3B, or control IgG) were added to the supernatant and incubated overnight at 4°C. The next day, the supernatant was incubated with Protein G PLUS/Protein A-Agarose (Millipore, IP10) and incubated for 2 h. Last, the complexes were analyzed using western blotting following the method described above.

LDL uptake in HUVECs

Cells were cultured in confocal dishes and incubated with serum-free OptiMEM (Gibco, 31,985,070) containing DiI-LDL (50 μg/mL; Yiyuan Biotechnology, YB-0011) for 3 h at 37°C. The cells were washed 3 times with PBS (Servicebio, G4202) and fixed with 4% paraformaldehyde for 20 min. Subsequently, the cell nuclei were stained using DAPI, and images of cells were acquired using confocal microscopy. (Olympus, FV3000).

LDL transcytosis

Fluorescein isothiocyanate (FITC)-LDL was prepared following a previously described method [15], and uncoupled FITC was removed by dialysis with PBS. HUVECs were cultured on Transwell membranes (CORNING, 3413) placed in 24-well plates, and the integrity of cellular monolayer was assessed by measuring transepithelial resistance (TEER) on a daily basis using a voltohmmeter (Millicell-ERS, Millipore, USA) until a stable value was achieved for 2 consecutive days [68,69]. After the incorporation of FITC-dextran (MW 4000; Beyotime, ST2930) into each transwell inserts, the fluorescence intensity in the lower wells were measured to exclude cells with more than 5% paracellular leakage. Subsequently, following a thorough washing process, the transendothelial amount of LDL was determined after treating cells with FITC-LDL (50 μg/mL). The fluorescence intensity of FITC was measured using fluorescence spectrophotometry (EnSpire, PerkinElmer) at excitation and emission wavelengths of 490 nm and 520 nm, respectively.

Immunofluorescence (IF) analysis

HUVECs were cultured in confocal dishes and given specific treatment, then fixed with 4% paraformaldehyde for 30 min at room temperature. After treatment with Immunostaining Permeabilization Buffer with Saponin (Beyotime, P0095) and Blocking Buffer for Immunol Staining (Beyotime, P0260) for 10 min each, the cells were incubated overnight with the primary antibodies (anti-CAV1 and anti-LC3B) at 4°C. The following day, the cells were incubated with the secondary antibodies and then with DAPI. Images of stained cells were acquired using confocal microscopy (Olympus, FV3000).

Transmission electron microscopy

After treatment, HUVECs were fixed overnight with 2% glutaraldehyde at 4°C, washed with PBS, and post-fixed with 1% osmium tetroxide (OSO₄) for 2 h. After washing again, the cells were dehydrated by adding different concentrations of ethanol (30%-100%). Last, the completely dehydrated cells were embedded in Embed-812 (EMS, 14,120). The specimens were dried and ultrathin sections were prepared using an automatic ultramicrotome (Leica, Germany). Next, the sections were stained with uranyl acetate and lead citrate, observed using TEM (FEI Tecnai G2 F20 S-TWIN), and photographed for documentation.

Immunoelectron microscopy

After treatment, HUVECs were fixed in 4% paraformaldehyde at 4°C for 2 h, infiltrated with 0.1% Triton X-100 (Servicebio, G3068) for 15 min, and finally blocked with serum (Servicebio, G8003) for 30 min. After 3 washes with PBS, the cells were incubated overnight with the primary anti-CAV1 antibody at 4°C and then with 5-nm colloidal gold-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, 115–185–146) for 24 h and washed with PBS. Next, the cells were continuously fixed with 2.5% glutaraldehyde at 4°C until they were embedded with Embed-812. After 48 h of polymerization at 60°C, ultrathin sections were obtained using a Leica ultramicrotome and placed on Formvar film/coated copper grids (EMS, ga300 Cu). The prepared cells were examined, and images were acquired using an HT7700 transmission electron microscope (HITACHI, Tokyo, Japan).

Animal experiments

All mice experiments were in accordance with the ARRIVE 2.0 guidelines [70]. Forty 4-week-old apoe^-/- male mice under the C57BL/6J genetic background were purchased from Vital River Technology Company (Beijing, China). After a week of adapted feeding of a standard diet, the mice were randomized into 2 groups. Each group of mice was intraperitoneally injected with 50 mg/kg streptozotocin (STZ; Shanghai Yisheng, 60256ES60; n = 20) or an equivalent amount of sodium citrate (n = 20) daily for 5 days. The successful establishment of the mouse model of diabetes was confirmed based on fasting blood glucose (FBG) levels > 16.7 mmol/L [67]. The diabetic and nondiabetic mice were randomized into 2 groups and injected with Ad-IMD-CBM (n = 10) or Ad-Vehicle (n = 10) at a dose of 8 × 10⁹ PFU through the tail vein every 2 weeks. After 14 weeks of being fed a high-fat diet (HFD, 42% kcal carbohydrates, 41% kcal fat, and 17% kcal protein; HFK Bioscience, H10141), the mice were euthanized with CO₂.

After anesthesia, the eyeballs were removed, and the blood was collected. The aorta and heart were isolated and fixed in 4% paraformaldehyde. The aortic root and the entire aorta were stained with Oil Red O (Servicebio, G1015), and the area occupied by the atherosclerotic plaques was quantified using ImageJ. Aortic cross-section rings were utilized in this study to determine the expression of CAV1, SQSTM1/p62, and LC3B. Immunohistochemistry was used to determine protein expressions, and TEM was used to observe the autophagic vacuoles present in the arteries.

Statistical analysis

Data are presented as mean ± SD. Student’s t-test was used for comparisons between 2 groups. One-way ANOVA followed by the Bonferroni correction was used for comparing multiple groups. p < 0.05 was established to determine statistical significance.

Funding Statement

The work was supported by the National Key Research and Development Program of China [2020YFC2008900]; National Natural Science Foundation of China [82070862, 82370840].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Supplementary material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/15548627.2026.2631946