Angiotensin-(1-7) promotes mitochondrial translocation of human telomerase reverse transcriptase in HUVECs through the TOM20 complex

Background: Angiotensin (Ang) (1-7) is a vasodilator peptide that ameliorates microcirculation dysfunction, increases Telomerase activity in cells, and exerts vasodilatory, anti-inflammatory, antioxidative stress, and antiapoptotic effects. Mitochondrial human telomerase Reverse Transcriptase (hTERT) plays an important role in the processes of antiapoptosis, antioxidative stress, and immortalization. This study aimed to investigate the effect of Ang(1-7) on the mitochondrial translocation of hTERT.

Methods: An in vitro model of lipopolysaccharide (LPS)-induced inflammation was established in human umbilical vein endothelial cells (HUVECs). Ang(1-7) was added to cells 30 min before LPS stimulation. The Ang(1-7)/Mas receptor antagonist A779 plus Ang(1-7) were added to the cells 30 min before LPS stimulation. The translocase outer membrane (TOM)20-overexpression HUVECs (HUVEC-TOM20^OE), TOM20-knockdown HUVECs (HUVEC-TOM20^KD), and the corresponding negative control cell lines were constructed by lentiviral transfection of HUVECs. Cells subjected to LPS stimulation alone, LPS plus Ang(1-7), LPS plus Ang(1-7) and A779, vehicle and no treatment were termed the LPS group, LPS + A group, LPS + A + A779 group, Con group and Neg group, respectively. Immunofluorescence staining was used to detect the distribution of hTERT in the nuclei and mitochondria of HUVECs and to locate TOM20, TOM40, and translocase inner membrane (TIM)23 in the mitochondria. The protein expression levels of total hTERT, mitochondrial hTERT, TOM20, TOM40, and TIM23 were measured by Western blot. The mRNA expression levels of hTERT, TOM20, TOM40, and TIM23 were assessed by quantitative reverse transcription-polymerase chain reaction (qRT-PCR).

Results: hTERT colocalized with TOM40, TOM20 and TIM23 in the mitochondria. The mitochondrial hTERT protein level of the LPS + A group was significantly greater than that of the LPS group (P = 0.001), and the LPS group showed significantly increased expression of mitochondrial hTERT compared with that of the control group (P = 0.001). No significant difference in the level of total hTERT was observed between the LPS + A and LPS groups. The mitochondrial hTERT protein level of the LPS + A + A779 group was significantly lower than that of the LPS + A group (P = 0.021). The protein level of mitochondrial hTERT in HUVEC-TOM20^KD treated with or without LPS alone or LPS + A was significantly decreased compared with the corresponding groups of control HUVECs (HUVEC-TOM20^KD-Con vs. HUVEC-Con, P = 0.035; HUVEC-TOM20^KD-LPS vs. HUVEC-LPS, P = 0.003; HUVEC-TOM20^KD-LPS + A vs. HUVEC-LPS + A, P = 0.001), and treatment with Ang(1-7) did not restore the downregulation of mitochondrial hTERT in HUVEC-TOM20^KD. HUVEC-TOM20^OE showed a significantly increased level of mitochondrial hTERT (HUVEC-TOM20^OE-Con vs. HUVEC-Con, P = 0.010), which was further elevated by Ang(1-7) stimulation (HUVEC-TOM20^OE-LPS + A vs. HUVEC-TOM20^OE-Con, P = 0.011). Lastly, the protein expression levels of TOM40 (HUVEC-TOM20^KD-Con vs. HUVEC-Con, P = 0.007) and TIM23 (HUVEC-TOM20^KD-Con vs. HUVEC-Con, P = 0.001) were significantly increased in HUVEC-TOM20^KD in comparison to HUVECs.

Conclusions: Ang(1-7) effectively promoted mitochondrial translocation of hTERT in HUVECs via TOM20, indicating that hTERT may be transported to the mitochondria through the TOM20 complex. In addition, A779 could block the effects of Ang(1-7) in HUVECs.