Protein disulfide-isomerase A3 knockdown attenuates oxidized low-density lipoprotein-induced oxidative stress, inflammation and endothelial dysfunction in human umbilical vein endothelial cells by downregulating activating transcription factor 2
Authors: Qiurong Yang
Ruijuan Huang
Xiaozhu Shen
Fengxia Du
Juan Li
Yueping Wang
Jing Jia
Journal: Bioengineered (2022)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: ATF2; PDIA3; atherosclerosis; inflammation; oxidative stress.
Affiliations: Department of Anesthetic Surgery, Baotou Steel Hospital, Baotou, China, Department of Cardiology, Baotou Steel Hospital, Baotou, China, Laser Treatment Center, Baotou Steel Hospital, Baotou, China, Department of Intensive Medicine, Baotou Steel Hospital, Baotou, China, Department of Geriatrics, The Second People’s Hospital of Lianyungang, Lianyungang, Jiangsu, China, Nursing Department, The Second People’s Hospital of Lianyungang, Lianyungang, Jiangsu, China

Abstract:  Atherosclerosis is a chronic inflammatory disease implicated in oxidative stress and endothelial dysfunction. Protein disulfide-isomerase A3 (PDIA3) has been reported to regulate oxidative stress and suppress inflammation. This study aimed to explore the function of PDIA3 in atherosclerosis and the underlying mechanisms. PDIA3 expression in oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) was detected using RT-qPCR and Western blotting. Following PDIA3 knockdown through transfection with small interfering RNA targeting PDIA3, cell viability, oxidative stress and inflammation in ox-LDL-induced HUVECs was examined using a Cell Counting Kit-8, corresponding kits and ELISA, respectively. The levels of CD31, α-smooth muscle, iNOS, p-eNOS, eNOS and NO were assessed using RT-qPCR, Western blotting and an NO kit to reflect endothelial dysfunction in ox-LDL-induced HUVECs. The relationship between PDIA3 and the activating transcription factor 2 (ATF2) was confirmed using co-immunoprecipitation. In addition, ATF2 expression was examined following PDIA3 silencing. The results indicated that PDIA3 was highly expressed in ox-LDL-induced HUVECs. PDIA3 silencing increased cell viability, and reduced oxidative stress and inflammation, as evidenced by the decreased levels of reactive oxygen species, malondialdehyde, TNF-α, IL-1β and IL-6, and increased superoxide dismutase and glutathione peroxidase activity. In addition, PDIA3 deletion improved endothelial dysfunction. PDIA3 interacted with ATF2, and PDIA3 deletion downregulated ATF2 expression. Furthermore, ATF2 overexpression reversed the effects of PDIA3 knockdown on ox-LDL-induced damage of HUVECs. Collectively, PDIA3 knockdown was found to attenuate ox-LDL-induced oxidative stress, inflammation and endothelial dysfunction in HUVECs by downregulating ATF2 expression, showing promise for the future treatment of atherosclerosis. 

Retracted: No

PubPeer comments:
 Fig 5.  Flow cytometric quantification of rate of apoptosis after treatment with maslinic acid against neuroblastoma cells at indicated doses. Results indicated that percentage apoptosis increased potentially on increasing the maslinic acid concentrations as indicated. All the data indicated in this figure are represented as meanu2009u00b1u2009SD and pu2009 u20090.05 was considered as statistically significant . ![file] For comparison: *[Left]* Figure 4 from [Lu et al (2019)] .  Annexin V PI staining showing the percentage of apoptotic MDA-T32 cells at each phase of cell cycle. The figure depicts that ferruginol increased the percentage of the apoptotic cells in a concentration dependent manner . *[right]* Figure 5 from [Yang et al (2018)] .  Estimation of apoptotic A375 populations at indicated doses by annexin V PI using flow cytometric analysis. The images are representatives of three biological experiments. The Figure indicates that the fraction of apoptotic cells (both early and late apoptosis) increased considerably with the increasing concentrations of KAM. ![] Other appearances are linked in those threads. , 
 Fig. 9  Images presenting the cellular invasion of SHSY-5Y cells after being exposed to maslinic acid at indicated doses . I am not convinced that the 0- and 10-microM panels emerged from differently-treated samples. ![file] , 
 *[lower left]* Fig. 8 from [Liu et al (2020)] .  Images presenting the cellular migration of SHSY-5Y cells after being exposed to maslinic acid at indicated doses. 0- and 10-microM panels overlap, as shown by orange boxes. *[upper left]* Figure 9 from [Yu, Fei Qin (2020)] .  Transwell chambers assay results presenting the number of migrated OVACAR-3 cells after u03b1-mangostin exposure. The figure demonstrates reduced number of migrated cells post u03b1-mangostin treatment . *[lower right]* Fig. 9 from [Yang et al (2020)] .  Transwell chambers assay results revealing the number of invasive HeLa cells after garcinone-E exposure . 16-microM panel is an enlarged section of the 0-microM panel, after a 180-deg rotation, as shown by brown boxes. Red, blue, green and yellow boxes indicate overlaps among the three papers. ![] , 
 *[left]* Fig. 4.  A concentration of 4u00d7105 cells was loaded to 24-well plates and maslinic acid treatment was performed for 24 h at indicated doses. Results revealed early and late apoptotic, and necrotic cellular morphology through fuorescence microscopy and represented by arrows. *[right]* Figure 2 from [Lin Ye (2020)] .  Acridine orange ethidium bromide (AO EB) staining of treated SKOV-3 OC cells showing chromatin condensation and nuclear fragmentation which increased with increasing dose of ursolic acid. Rotated through 90 deg for ease of comparison. Two overlaps are noted, but there appear to be selective local differences. ![file] , 
 *[below]* Fig 4 again. *[above]* Figure 2 from [Chen, Lu Hu (2019)] .  A: AO EB staining . ![file] ,