Advanced glycation end products promote the release of endothelial cell-derived mitocytosis

Abstract

Accumulation of advanced glycation end products (AGEs) and endothelial dysfunction are major factors that contribute to the progression of vascular complications in diabetes. Migrasomes, a newly discovered organelle involved in mitocytosis, play an important role in the selective removal of damaged mitochondria. Our research shows that human umbilical vein endothelial cells (HUVECs) can release migrasomes and undergo mitocytosis. In addition, when exposed to oxidative stress from AGEs, mitochondrial damage worsens, leading to the activation of migrasome-mediated mitocytosis. We also found that migrasomes carrying mitochondria can be taken up by recipient cells. Understanding the connection between migrasome release, mitocytosis, and mitochondrial function in endothelial cells sheds light on the biological processes behind intercellular communication.

Keywords: AGEs; HUVECs; diabetes; migrasomes; mitocytosis.

Fig. 1

HUVECs release migrasomes and exhibit mitocytosis. (A) Colocalization of TSPAN4 with WGA in HUVECs. HUVECs were transfected with LV8N‐TSPAN4 and immunofluorescently labeled for Flag (red) and migrasomes (green), n = 3. Scale bar = 81.9 μm. The lower panels depict an enlarged ROI with a scale bar = 20 μm. (B) TEM reveals migrasomes and mitocytosis released by HUVECs, n = 3. In the upper panels, the left Scale bar = 5 μm and the right Scale bar = 1 μm. The lower panels depict an enlarged ROI with a scale bar = 500 nm. (C) Confocal image of HUVECS stained with WGA, MitoTracker, n = 3. Scale bar = 81.9 μm; The right panels, an enlarged ROI is shown with a scale bar of 20 μm. HUVECs, human umbilical vein endothelial cells; ROI, region of interest; TEM, transmission electron microscopy; TSPAN4, tetraspanin‐4; WGA, wheat‐germ agglutinin.

Fig. 2

AGEs induce mitochondrial damage and promote migrasome‐mediated mitocytosis in HUVECs. (A) HIUVECs were treated with AGEs, leading to oxidative stress, n = 3/group. Scale bar = 70 μm. The graph on the right shows the relative fluorescence intensity of DCFH‐DA. (B) Mitochondrial membrane potential of HIUVECs was treated with AGEs, n = 3/group. Scale bar = 250 μm. The graph on the right shows the relative fluorescence intensity of JC‐1 monomers. (C) Mitochondrial oxidative stress of HIUVECs was treated with AGEs, n = 3/group. Scale bar = 250 μm. The graph on the right shows the relative fluorescence intensity of MitoSOX Red. (D) Representative TEM results show mitophagy in HUVECs incubated with AGEs or control AGEs for 24 h, n = 3/group. Red arrowheads indicate mitochondrial disruption and mitophagy‐like structures. Scale bar (left) = 2 μm; scale bar (right) = 500 nm. (E) Representative confocal image taken after HUVECs were treated with AGEs for 24 h, stained with WGA and MitoTracker, n = 3/group. Scale bar = 81.9 μm. The graphs on the right display the statistical count of migrasomes per cell and the number of mitocytosis events per cell. (F) TEM was used to observe migrasomes and mitochondria within the migrasomes, with red arrowheads highlighting migrasome‐mediated mitocytosis, n = 3/group. Scale bar = 1 μm; for the right panels, an enlarged ROI is shown with a scale bar of 500 nm. AGEs, advanced glycation end products; TEM, transmission electron microscopy; WGA, wheat‐germ agglutinin. Data in A–C and E are quantified with three random fields for each sample. The error bars represent the mean ± SEM (standard error of the mean) with the indicated significance. Statistical analysis utilized Student's t‐test (ns, no significance; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Fig. 3

Characterization and uptake of migrasomes isolated from HUVECs. (A) Schematic representation of the isolation process for migrasomes derived from cultured HUVECs. (B) Electron microscopic picture of migrasomes, n = 3. Left image scale bar = 500 nm; Right image scale bar = 200 nm. (C) Western blot (WB) analyses confirm the presence of specific markers within the extracted migrasomes. Sample 1–3 represent three independent replicates of western blot analysis for both CM and AM groups. CM represents CON migrasomes; AM represents AGEs‐treated migrasomes. (D) Schematic illustration of co‐culture experiments involving HUVECs and migrasomes. (E) Coculture of migrasomes (Red) and HUVECs (Green), with images captured via confocal microscopy to observe the transfer of migrasomes (Mig) to HUVECs, n = 3. Scale bar = 10 μm. The lower panels depict an enlarged ROI with a scale bar = 2.5 μm. Fluorescent markers: Red—DiR; Green—DiO; Blue—Hoechst. (F) Confocal microscopy images illustrating the transfer of mitochondria within migrasomes (mt‐Mig) (Red) to HUVECs (Green), n = 3. Scale bar = 10 μm. The lower panels depict an enlarged ROI with a scale bar =2.5 μm. Fluorescent markers: Red—MitoTracker; Green—DiO; Blue—Hoechst.