Fig 2: MiR-505 promotes endothelial activation. (A) Agomir-505-transfected and agomir-NC-transfected HUVECs were exposed to vehicle (VC) or 2 ng/mL TNF-α for 16 h, followed by real-time qPCR analysis of the expression of ICAM1, VCAM1 and E-selectin. Relative fold change of gene expression was plotted against that from agomir-NC-transfected vehicle-treated cells (n = 6 per group). Data represent mean ± SEM. P values are derived from one-way ANOVA and the Student’s t-test. (B) Antagomir-505-transfected and antagomir-NC-transfected HUVECs were treated with vehicle or 2 ng/mL TNF-α for 16 h, followed by real-time qPCR analysis of the expression of ICAM1, VCAM1 and E-selectin. Relative fold change of gene expression was plotted against that from antagomir-NC-transfected vehicle-treated cells (n = 6 per group). Data represent mean ± SEM. P values are derived from one-way ANOVA and the Student’s t-test. (C) Agomir-505, antagomir-505 and their respective negative controls were transfected in HUVECs, followed by vehicle or 2 ng/mL TNF-α treatment for additional 16 h. The protein level of VCAM1 was then examined by western blotting. Relative fold change of VCAM1 protein was plotted against that from agomir-NC-transfected TNF-α-stimulated cells or antagomir-NC-transfected TNF-α-stimulated cells (n = 4 per group). Data represent mean ± SEM. P values are derived from the Student’s t-test. (D) HUVECs were transfected with agomir-505, antagomir-505 and their respective negative controls, followed by vehicle or 2 ng/mL TNF-α treatment for 12 h. The culture medium was then collected and analyzed for the level of sE-selectin by ELISA (n = 6 per group). Data represent mean ± SEM. P values are derived from the Student’s t-test. (E) Agomir-505, antagomir-505 and their respective negative controls were transfected in HUVECs, followed by vehicle or 2 ng/mL TNF-α treatment for additional 16 h. The adhesion of CMFDA-labeled THP-1 cells to HUVECs was then examined by measuring the fluorescence intensity of CMFDA. Relative fold change of adhesion was plotted against that from agomir-NC-transfected vehicle-treated cells or antagomir-505-transfected vehicle-treated cells (n = 6 per group). Data represent mean ± SEM. P values are derived from one-way ANOVA and the Student’s t-test.

Fig 3: HSP22 reduces diabetes-induced endothelial injuryin vivo. Diabetes was induced in wild-type (WT) and transgenic (TG) mice by HFD adjuvanted with STZ injection for 12 weeks (n = 8 per group). A) Blood glucose concentrations in the WT-control, TG-control, WT-diabetic and TG-diabetic mice were measured at baseline and 4, 8, and 12 weeks after diabetes induction. B) The body weights of the WT-control, TG-control, WT-diabetes and TG-diabetes mice were measured at baseline and 4, 8, and 12 weeks after diabetes induction. C) Representative images of H&E staining (original magnification ×40). D) Representative immunohistochemical staining images of HSP22, ICAM-1 and VCAM-1 in aortic sections (original magnification ×40). E) Quantification of positive staining for HSP22 in aortic sections. n = 5. F) mRNA expression of HSP22 in the aorta. G) Quantification of positive staining for ICAM-1 and VCAM-1 in aortic rings. n = 5. H) mRNA expression of IL5, IL6, IL13, Mip3α and TGFβ1 in the aorta. *P < 0.05 vs. WT-control; #P < 0.05 vs. WT-diabetes；&P < 0.05 vs. TG-control.

Fig 4: Autophagy contributes to TNFα-induced VCAM1 activation. (A) Relative mRNA level of VCAM1. (B) VCAM1 protein expression and the quantification data; EA.hy926 cells were pretreated with rapamycin for 2 h, followed by 12 h TNFα stimulation. (C) Relative VCAM1 gene expression level, (D) VCAM1 protein expression level and the quantification data; EA.hy926 cells were treated with si ATG5 or scrambled siRNA (si NC) for 24 h and then incubated with TNFα or vehicle for another 12 h. All values are denoted as the means ± SD from at least three independent tests. Each group contains the same amount of solvent. ud, undetectable. ∗p < 0.05 and ∗∗p < 0.01 indicate statistically significant differences.

Fig 5: Hyperglycemia induces HSP22 upregulation and endothelial activationin vitro. Human umbilical vein endothelial cells (HUVECs) were cultured in endothelial basal medium (EBM) containing normal glucose (NG, 5 mM D-Glucose), osmotic control (OC, 30 mM D-mannitol), or high glucose (HG, 30 mM D-Glucose) for 24 h. A) Cell viability was evaluated in cells in 96-well plates by a Cell Counting Kit-8 (CCK8) after treatment with NG, OC or HG for 24 h. n = 3. B) HUVECs were cultured in EBM containing NG, OC or HG for 24 h, and the cell media were collected for the measurement of lactate dehydrogenase (LDH) release as an indicator of cytotoxicity. n = 3. C) HUVECs were cultured in EBM containing NG, OC or HG for 24 h, and the adhesion of nonstimulated primary human peripheral mononuclear cells (PBMCs) to stimulated HUVECs was quantified. n = 3. D) mRNA expression of ICAM-1 and VCAM-1 was determined by RT-PCR. n = 3. E) Endothelial cell activation-related cytokines were examined by RT-PCR. n = 3. F) Representative immunofluorescence staining images of HSP22 (green), the endothelial cell marker CD31 (red), and nuclei (blue) (original magnification ×63). G) Quantification of the fluorescence intensity of HSP22 and CD31 in HUVECs. n = 3. H) mRNA expression of HSP22 was determined by RT-PCR. n = 3. I) HSP22 protein expression was determined by Western blotting. J) Quantification of HSP22 protein expression. GAPDH was used as a loading control. n = 3. *P < 0.05 vs. NG; #P < 0.05 vs. OC.