Figure 5.

EVA1A (eva-1 homolog A) promotes endothelial apoptosis via regulation of autophagy. A and B, Human umbilical vein endothelial cells (HUVECs) were treated with EVA1A siRNA (small interfering RNA) or with nontargeting control (NTC) siRNA before exposing to flow for 72 h using the orbital shaker system. To block autophagic flux, ECs were treated with 50 nM bafilomycin for the last 4 h of flow exposure. Control cells were treated with 0.05% dimethyl sulfoxide (DMSO). EC apoptosis under disturbed flow (DF) was assessed by immunostaining using antiactive caspase 3 antibody (A, green) and costaining of nuclei with DAPI (4′,6-diamidino-2-phenylindole; A, blue). Apoptotic ECs are indicated with white arrows. The graph in B represents percentage of apoptotic ECs calculated by dividing the number of active caspase 3–positive cells by the total number of ECs per field of view (n=3). C, Human aortic ECs (HAECs) were treated with TWIST1 (twist basic helix-loop-helix transcription factor 1) siRNA or with NTC siRNA before exposing to flow for 72 h using the ibidi parallel plate system. The ECs were exposed to either high laminar shear stress (HSS) of 13 dynes/cm², low laminar shear stress (LSS) of 4 dynes/cm², or low oscillatory shear stress (LOSS) of 4 dynes/cm² with 1 Hz oscillations. Expression of EVA1A mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR; n=3 donors), using HPRT as a housekeeping gene. B and C, Data are presented as means±SE of the mean and normalized to control (C). Differences between groups were analyzed using a 2-way ANOVA with Tukey post hoc test and P values are shown in the graphs. D, A schematic diagram summarizing proposed mechanism of how EVA1A (Continued )Figure 5 Continued. regulates endothelial function under different hemodynamic conditions. Under DF conditions, EVA1A expression is upregulated in ECs and contributes to the DF-induced reduction in autophagic flux, leading to increased EC apoptosis and inflammatory activation, which results in EC dysfunction and lesion development (this study). EVA1A expression can also be induced in injured ECs under undisturbed flow (UF) conditions, where it promotes EC repair by regulating EC proliferation and migration via RAC1/CDC42/ARPC1B (rac family small GTPase 1/cell division cycle 42/actin-related protein 2/3 complex subunit 1B) pathway (study by Li et al²⁰). Scale bar: (A), 100 µm.