ER-Stress and Senescence Coordinately Promote Endothelial Barrier Dysfunction in Diabetes-Induced Atherosclerosis

Figures

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  Figure 1. Hyperglycaemia-induced senescence promotes barrier disruption in endothelial cells. (A) Experimental design and treatment conditions. (B,C) Dot plot summarizing data of barrier integrity measured by FITC dextran leakage (B) and TEER (C). (D–G) Exemplary immunoblot of p21, p16, and p53 ((D), loading control: GAPDH) and dot plot summarizing p21 (E), p16 (F) and p53 (G) densitometric quantifications of immunoblots. (H–L) Representative immunofluorescence images showing endothelial cell staining for p21 ((H), top panel, red; nuclear counterstain: DAPI, blue), p16 ((H), first middle panel, red; nuclear counterstain: DAPI, blue), p53 ((H), second middle panel, red; nuclear counterstain: DAPI, blue) and senescence-associated beta galactosidase staining ((H), bottom panel, blue). Dot plots summarizing data for p21 (I), p16 (J), p53 (K) and SAβ gal (L). Scale bar: 20 µm (H). HCAEC maintained under control (non-treated, C), high glucose (25 mM; HG) or oxidized low density lipoprotein (oxLDL 50 µg/mL, oxLDL) conditions. Each dot represents data obtained from one biological specimen; * p < 0.05, ** p < 0.01; ANOVA.

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  Figure 2. Senescence is induced within plaques of diabetic ApoE−/− mice. (A) Representative immunofluorescence images showing staining of brachiocephalic arteries for p21 ((A), top panel, p21, green; nuclear counterstain: DAPI, blue), p16 ((A), middle panel, p16, red; nuclear counterstain: DAPI, blue) and p53 ((A), bottom panel, p16, red; nuclear counterstain: DAPI, blue). (B–D) Dot plots summarizing immunofluorescence data for p21 (B) p16 (C), p53 (D) and Scale bar: 20 µm (A). ApoE−/− control mice (Cont, normal chow diet, citrate instead of streptozotocin injections), DM mice (normal chow diet, streptozotocin injections) or HFD mice (fed high fat diet). Each dot represents data obtained from one mouse specimen; ** p < 0.01; ANOVA.

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  Figure 3. Hyperglycaemia induces UPR in endothelial cells and within plaques of diabetic ApoE−/− mice. (A–C) Representative immunoblots spliced XBP1 (sXBP1), total ATF6, and cleaved ATF6 alpha (cl-ATF6α) ((A), loading control: GAPDH). Dot plots summarize densitometric quantifications of immunoblotting results for sXBP1 (B) and cl-ATF6α (C). (D–F) Representative immunofluorescence images showing endothelial cells staining for sXBP1 ((D), upper panel, red; nuclear counterstain: DAPI, blue) and cl-ATF6-α ((D), lower panel, red; nuclear counterstain: DAPI, blue). Dot plots summarizing immunofluorescence data for sXBP1 (E) and cl-ATF6-α (F). Scale bar: 20 µm (D). (G–I) Representative immunofluorescence images showing staining of brachiocephalic arteries for sXBP1 ((G), upper panel, sXBP1, red; DAPI nuclear counterstain, blue) and cl-ATF6-α ((G), lower panel, cl-ATF6-α, green; DAPI nuclear counterstain, blue). Dot plots summarizing immunofluorescence data for sXBP1 (H) and cl-ATF6-α (I). Scale bar: 20µm (G). HCAECs maintained under control (C), high glucose (25 mM; HG) or oxidized low density lipoprotein (oxLDL 50 µg/mL, oxLDL) conditions. Each dot represents data obtained from one biological…

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  Figure 4. aPC avert glucose-induced UPR. (A–C) Representative immunoblots showing spliced XBP1 (sXBP1), total ATF6 and cleaved ATF6 alpha (cl-ATF6α) ((A), loading control: GAPDH). Dot plots summarize densitometric quantifications of immunoblotting results for sXBP1 (B) and cl-ATF6α (C). (D–F) Representative immunofluorescence images showing staining for sXBP1 ((D), middle panel, red; nuclear counterstain: DAPI, blue) and cl-ATF6-α ((D), bottom panel, red; nuclear counterstain: DAPI, blue). Dot plots summarizing immunofluorescence data for sXBP1 (E) and cl-ATF6-α (F). Scale bar: 20 µm (D). HCAECs maintained under control (C), high glucose (25 mM; HG) or HG + aPC (25 mM glucose + 20 nM of exogenous activated protein C) conditions. Each dot represents data obtained from one biological specimen. ** p < 0.01; ANOVA.

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  Figure 5. aPC reduces glucose-induced senescence. (A–C) Representative immunoblots showing p21 and p16 expressions ((A), loading control: GAPDH). Dot plots summarize densitometric quantifications of immunoblotting results for p21 (B) and p16 (C). (D–F) Representative immunofluorescence images showing endothelial cells staining for p21 ((D), upper panel, p21, red; nuclear counterstain: DAPI, blue) and p16 ((D), lower panel, p16, red; nuclear counterstain: DAPI, blue). Dot plots summarizing immunofluorescence data for p21 (E) and p16 (F). Scale bar: 20 µm (D). (G,H) Dot plot summarizing barrier integrity data measured by FITC dextran leakage (G) and TEER (H). HCAECs maintained under control (C), high glucose (25 mM; HG), or HG+aPC (25 mM glucose + 20nM of exogenous activated protein C) conditions. Each dot represents data obtained from one biological specimen. ** p < 0.01; ANOVA.