Stacked human aortic endothelial cells induce atherosclerotic fatty streaks and release proinflammatory cytokines and chemokines

Figures

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  Fig. 1. Origin of fatty streaks and their formation from coralthelial cells transformed from stacked human aortic endothelial cells (HAECs). (A) Schematic of the traditional and current views regarding fatty streak formation. Traditional view (left): Circulating monocytes are recruited by inflammatory chemokines to sites of endothelial damage, where they enter the intima along with fluid, lipids, and other circulating substances. The accumulated low-density lipoprotein (LDL) in the intima can undergo oxidation and be avidly taken up by monocyte-derived macrophages and vascular smooth muscle cells (VSMCs) migrating into the intima from the media to form macrophage-derived and VSMCs-derived foam cells, respectively. The fatty streak–like structure comprises these lipid-containing foam cells in the arterial wall beneath the endothelium. Current view (right): The initial lipid deposition occurs in the deeper intimal layers proximal to the internal elastic lamina, not in the leaky endothelium or region dominated by macrophage-derived foam cells. The intima covered by the regenerated endothelium is considerably thicker and more likely to accumulate lipids. Surprisingly, approximately 25% or…

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  Fig. 2. Ultrastructure of coralthelial cells, nuclear import of Golgi, lipid deposition, and endothelial marker status. (A) After culturing monolayer HAECs and coralthelial cells for 11 days, their ultrastructure and cytoplasmic organelles were assessed by TEM. Size quantification of cells (B), nuclei (C), mitochondria (D), autophagic vacuoles (E), and lipid droplets (F). (G) Ultrastructure and location of the Golgi apparatus in coralthelial cells. (H) Monolayer HAECs and coralthelial cells were co-stained with anti-GOLGA2/GM130 and -SAR1B antibodies. The MFIs of GM130 (I) and SAR1B (J) in the nucleus, cytoplasm, and overall are presented as fold-changes relative to their MFIs in the cytoplasm per cell in the HAEC monolayer. Lipid droplet deposition was assessed by staining with Oil Red O (K) and BODIPY lipid probes (M). (L) and (N) show the corresponding positive staining area (PSA) in terms of percentage (%) or MFI. Cells were also stained for the endothelial markers CD31 (O) and FVIII (Q). (P) and (R) show the corresponding MFI values. The 3D views in the right top corner in (M, O, and Q) correspond to the red boxed regions in these panels. WP, Weibel–Palade body; V, pinocytic…

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  Fig. 3. RNA sequencing analysis of coralthelial cells and chemokines/cytokines detection. (A) Volcano plot of DEGs. The red and green spots indicate up- and down-regulated genes, respectively. The chemokines/cytokines IL-6, MCP-1, and CXCL8, which plays a role in human cardiovascular events, are marked in blue. (B) PCA based on the transcriptome profile indicates that the first principal component (PC1) can separate coralthelial cells (stack) from HAECs (monolayer). (C) GO analysis of DEGs indicates that the significantly enriched GO terms in coralthelial cells are related to chemokine/cytokine signaling in inflammation. (D) Pathway analysis of DEGs corroborates that chemokine/cytokine signaling pathways in inflammation are significantly altered in coralthelial cells. (E–G) qRT-PCR confirms increased mRNA expression of IL-6 (E), MCP-1 (F), and CXCL8 (G) in coralthelial cells. (H–J) ELISA detection of IL-6 (H), MCP-1 (I), and CXCL8 (J) in the culture medium of coralthelial cells and that of HAECs on days 2, 5, 8, and 11. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 vs. HAECs (monolayer).

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  Fig. 4. RPL23 translocates into the nucleolus and facilitates the secretion of proinflammatory cytokines, which is attenuated by SAR1B siRNA. (A) Co-staining of RPL23 and FBL (the nucleolus marker) in monolayer HAECs and coralthelial cells. (B, C) Following SAR1B knockdown, the nuclear translocation of RPL23 (B) and GM130 (C) were assessed in coralthelial cells. Line scans were performed for RPL23 and FBL. The MFIs of RPL23, FBL, GM130, and SAR1B in the nucleus and cytoplasm are presented as fold-changes relative to their levels in the cytoplasm of monolayer HAECs or NC coralthelial cells (stack). Data are presented as the mean ± SD; ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001. (D) The levels of IL-6, MCP-1, and CXCL8 in coralthelial cells were quantified. ∗∗∗P < 0.001 vs. NC monolayer; ##P < 0.01, ###P < 0.01 vs. NC stack.

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  Fig. 5. Major differences between human aortic endothelial cells and coralthelial cells, and the mechanism of cytokine release in coralthelial cells. (A) HAECs display a typical endothelial morphology with a flattened, elongated structure, organized actin filaments, intact glycocalyx, and minimal lipid accumulation. (B) By contrast, coralthelial cells, derived from stacked HAECs, exhibit a distinct coral-like morphology with surface blebbing, smaller cell bodies and nuclei, disorganized actin filaments, degraded glycocalyx, smaller mitochondria, enhanced nucleolar stress, and prominent lipid droplet accumulation, resembling foam cells found in atherosclerotic plaques. Coralthelial cells also lack typical endothelial markers such as PECAM-1 (CD31) and FVIII, which are expressed in HAECs. Coralthelial cells show a marked increase in collagen production, helping form the extracellular matrix scaffold observed in atherosclerotic fatty streak–like structures. In addition, coralthelial cells exhibit significant upregulation of proinflammatory cytokines and chemokines, including IL-6, MCP-1 and CXCL8, promoting a proinflammatory microenvironment characteristic of atherosclerosis. This…