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Figure 1 | Molecular Medicine

Figure 1

From: Suppression of Coronary Atherosclerosis by Helix B Surface Peptide, a Nonerythropoietic, Tissue-Protective Compound Derived from Erythropoietin

Figure 1

HBSP and EPO inhibit HUVEC apoptosis via activation of molecular pathways increasing cell survival. (A) HBSP and EPO reduce CRP-induced HUVEC apoptosis. HUVECs were treated with vehicle, CRP (95.2 nmol/L), CRP + EPO (10,000 IU/L) or CRP + HBSP (2.0 or 20 nmol/L) for 24 h. Then cells were harvested by trypsinization, and flow cytometric analysis was performed to evaluate apoptosis. Data are mean ± SEM, n = 18 each group; *P < 0.01 versus CRP#P < 0.01 versus CRP + EPO. (B) HBSP and EPO activate Akt and ERK1/2. HUVECs were incubated with a control peptide (C; a scrambled version of HBSP 2.0 nmol/L, 15 min), HBSP (2.0 nmol/L) or EPO (10,000 IU/L) for the indicated times, and cell lysates were prepared. Next, Western blot was performed as described in Materials and Methods. Immunoreactive bands were analyzed by densitometry by using NIH ImageJ, and ratios of the phospho/total antibody immunolabeling were calculated. The ratios were compared between C and 15 min in HBSP and between 0 and 15 min in EPO. The pAkt/Akt ratios at 30 min in EPO were also calculated. Data are mean ± SEM, n = 3 each group; *P < 0.01, #P < 0.05. (C) A similar analysis performed after incubation in increasing concentrations of EPO or HBSP showed that a peak phosphorylation of Akt occurred at ~2 nmol/L. (D) Akt is required for HBSP-mediated inhibition of apoptosis. Akt siRNA, ERK siRNA or scrambled siRNA was transfected into HUVECs as described in Materials and Methods. Then apoptosis of HUVECs was evaluated as in (A). A, control + scrambled siRNA; B, CRP + scrambled siRNA; C, CRP + HBSP + scrambled siRNA; D, CRP + HBSP + Akt siRNA; E, CRP + HBSP + ERK siRNA. Data are mean ± SEM, n = 8 each group; *P < 0.05 versus CRP + scrambled siRNA, #P < 0.05 versus CRP + HBSP + scrambled siRNA and CRP + HBSP + ERK siRNA. (E) Akt is downregulated by Akt siRNA at the protein level. Akt siRNA or scrambled siRNA was transfected into HUVECs in the same manner as in (D), without CRP or HBSP treatment, and cell lysates were prepared. Next, Western blot was performed as described in Materials and Methods by using an antibody against Akt or β-actin (a loading control). Immunoreactive bands were analyzed by NIH ImageJ, and ratios of the Akt/β-actin antibody immunolabeling were calculated. Data are mean ± SEM, n = 3 each group; *P < 0.05.

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