Exosomal NEDD4L derived from HG+oxLDL-induced vascular endothelial cells accelerates macrophage M1 polarization and oxLDL uptake by ubiquitinating IκBα and PPARγ

Abstract

Background: Vascular endothelial cell-derived exosomes are thought to mediate disease progression by regulating macrophage polarization. However, its mechanism in diabetes mellitus (DM)-related atherosclerosis (AS) progress is unclear.

Methods: High-glucose (HG) and oxLDL were used to induce human cardiac microvascular endothelial cells (HCMECs) to mimic DM-related AS model. The conditioned medium (CM) from HG+oxLDL-induced HCMECs was incubated with THP1-M0 monocytes treated with LPS or oxLDL. The mRNA levels of macrophage M1/M2 polarization markers, NEDD4L, IκBα and PPARγ were determined by qRT-PCR. Flow cytometry was used to analyze macrophage marker. Dil-labeled oxLDL and oil red O staining were performed to assess oxLDL uptake by THP1-M0 cells. The levels of inflammatory factors were examined using ELISA. Transmission electron microscope was used for observing foam cell formation and exosome morphology. The protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα, p-P65/P65, anti-lipid metabolism-related markers, and NEDD4L were tested by western blot. The interaction between NEDD4L and IκBα or PPARγ was assessed by Co-IP assay.

Results: The CM of HG+oxLDL-induced HCMECs could promote macrophage M1 polarization, oxLDL uptake and foam cell formation, and exosome inhibiter GW4869 eliminated these effects. NEDD4L was overexpressed in exosomes from HG+oxLDL-induced HCMECs, which could be taken up by THP1-M0 cells. Exosomal NEDD4L knockdown inhibited macrophage M1 polarization, oxLDL uptake and foam cell formation by reducing the protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα and p-P65/P65. NEDD4L could reduce IκBα and PPARγ expression through ubiquitination.

Conclusion: HG+oxLDL-induced HCMECs-derived exosomal NEDD4L could enhance the ubiquitination of IκBα and PPARγ to facilitate macrophage M1 polarization and oxLDL uptake, thus accelerating DM-related AS.

Keywords: Atherosclerosis; Diabetes mellitus; Exosome; Macrophages; NEDD4L.

Fig. 1

Exosomes from HG+oxLDL-induced HCMECs mediated macrophage M1 polarization. A Flow chart of cell treatment and co-incubation. B-C qRT-PCR for detecting CD80, iNOS, IL-6, CD206, Arg-1 and IL-10 mRNA levels in LPS-induced THP1-M0 cells. D-E Flow cytometry for analyzing CD80 and CD206 positive cells in LPS-induced THP1-M0 cells. F-I ELISA was used to test the levels of IL-6, TNF-α, IL-1β and IL-10 in LPS-induced THP1-M0 cells. All experiments were performed in triplicate and P<0.05 was denoted statistically significant

Fig. 2

Exosomes from HG+oxLDL-induced HCMECs mediated oxLDL uptake and foam cell formation. A Flow chart of cell co-incubation. B Fluorescence microscope was used to observe Dil-labeled oxLDL in THP1-M0 cells. C Oil red O staining to evaluate lipid deposition in oxLDL-induced THP1-M0 cells. D TEM for observing foam cell formation. All experiments were performed in triplicate and P<0.05 was denoted statistically significant

Fig. 3

NEDD4L expression in exosomes from HG+oxLDL-induced HCMECs. A TEM for observing the microstructure of exosomes from HCMECs treated with Mannitol+nLDL or HG+oxLDL. B WB for testing TSG101, CD81, CD63 and Calnexin protein expression in exosomes from HCMECs treated with Mannitol+nLDL or HG+oxLDL. C NTA for detecting the particle sizes and concentrations of exosomes from HCMECs treated with Mannitol+nLDL or HG+oxLDL. D NEDD4L mRNA level was tested by qRT-PCR in HCMECs treated with Mannitol+nLDL or HG+oxLDL. E NEDD4L mRNA level in exosomes from HCMECs treated with Mannitol+nLDL or HG+oxLDL was measured by qRT-PCR. F Fluorescence microscope was used to observe Dil-labeled Exo in THP1-M0 cells after co-culturing with Exo^{Mannitol+nLDL} or Exo^HG+oxLDL. G-H qRT-PCR and WB for testing NEDD4L mRNA and protein levels in THP1-M0 cells after co-culturing with Exo^{Mannitol+nLDL} or Exo^HG+oxLDL. All experiments were performed in triplicate and P<0.05 was denoted statistically significant

Fig. 4

Effect of exosomal NEDD4L knockdown from HG+oxLDL-induced HCMECs on macrophage M1 polarization. A Flow chart of cell treatment and co-incubation. B-C qRT-PCR and WB for detecting NEDD4L mRNA and protein levels in LPS-induced THP1-M0 cells. D-E qRT-PCR for testing CD80, iNOS, IL-6, CD206, Arg-1 and IL-10 mRNA levels in LPS-induced THP1-M0 cells. F-G CD80 and CD206 positive cells were analyzed by flow cytometry in LPS-induced THP1-M0 cells. H-K The levels of IL-6, TNF-α, IL-1β and IL-10 in LPS-induced THP1-M0 cells was analyzed by ELISA. L Protein levels of p-Smad1/Smad1, p-Smad2/Smad2, p-IκBα/IκBα and p-P65/P65 in LPS-induced THP1-M0 cells were examined by WB. All experiments were performed in triplicate and P<0.05 was denoted statistically significant

Fig. 5

Effect of exosomal NEDD4L knockdown from HG+oxLDL-induced HCMECs on oxLDL uptake and foam cell formation. A Flow chart of cell co-incubation. B-C NEDD4L mRNA and protein levels in oxLDL-induced THP1-M0 cells were examined by qRT-PCR and WB. D Dil-labeled oxLDL in THP1-M0 cells was observed using fluorescence microscope. E Lipid deposition in oxLDL-induced THP1-M0 cells was assessed by Oil red O staining. F Foam cell formation was observed under TEM. G Protein levels of PPARγ, ABCG1, ABCA1 and LDLR in oxLDL-induced THP1-M0 cells were tested by WB. All experiments were performed in triplicate and P<0.05 was denoted statistically significant

Fig. 6

Effect of NEDD4L on the ubiquitination of IκBα and PPARγ. A-B The mRNA and protein levels of NEDD4L, IκBα and PPARγ were detected by qRT-PCR and WB in THP1-M0 cells transfected with si-NC/si-NEDD4L. C-D WB was used for detected IκBα and PPARγ protein levels in THP1-M0 cells transfected with si-NC/si-NEDD4L and treated with CHX for indicated time points. E-F Co-IP for assessing the interaction between NEDD4L and IκBα or PPARγ. G-H Detection of ubiquitination levels of IκBα and PPARγ in THP1-M0 cells transfected with si-NC/si-NEDD4L. All experiments were performed in triplicate and P<0.05 was denoted statistically significant

Fig. 7

The mechanism diagram of this study. Exosomal NEDD4L derived from HG+oxLDL-induced HCMECs could ubiquitinate IκBα/PPARγ and phosphorylate Smad1/Smad2, thus promoting macrophage M1 polarization, oxLDL uptake and foam cell formation