Elabela prevents angiotensin II-induced apoptosis and inflammation in rat aortic adventitial fibroblasts via the activation of FGF21-ACE2 signaling

Abstract

Apoptosis, inflammation, and fibrosis contribute to vascular remodeling and injury. Elabela (ELA) serves as a crucial regulator to maintain vascular function and has been implicated in the pathogenesis of hypertensive vascular remodeling. This study aims to explore regulatory roles and underlying mechanisms of ELA in rat aortic adventitial fibroblasts (AFs) in response to angiotensin II (ATII). In cultured AFs, exposure to ATII resulted in marked decreases in mRNA and protein levels of ELA, fibroblast growth factor 21 (FGF21), and angiotensin-converting enzyme 2 (ACE2) as well as increases in apoptosis, inflammation, oxidative stress, and cellular migration, which were partially blocked by the exogenous replenishment of ELA and recombinant FGF21, respectively. Moreover, treatment with ELA strikingly reversed ATII-mediated the loss of FGF21 and ACE2 levels in rat aortic AFs. FGF21 knockdown with small interfering RNA (siRNA) significantly counterbalanced protective effects of ELA on ATII-mediated the promotion of cell migration, apoptosis, inflammatory, and oxidative injury in rat aortic AFs. More importantly, pretreatment with recombinant FGF21 strikingly inhibited ATII-mediated the loss of ACE2 and the augmentation of cell apoptosis, oxidative stress, and inflammatory injury in rat aortic AFs, which were partially prevented by the knockdown of ACE2 with siRNA. In summary, ELA exerts its anti-apoptotic, anti-inflammatory, and anti-oxidant effects in rat aortic AFs via activation of the FGF21-ACE2 signaling. ELA may represent a potential candidate to predict vascular damage and targeting the FGF21-ACE2 signaling may be a promising therapeutic intervention for vascular adventitial remodeling and related disorders.

Keywords: Adventitial fibroblasts; Angiotensin-converting enzyme 2; Apoptosis; Elabela; Fibroblast growth factor 21; Inflammation.

Fig. 1

Regulatory roles of ELA in cell apoptosis and oxidative stress in rat aortic AFs. a Relative mRNA levels of ELA in rat aortic AFs in response to ATII, ELA, and ELA siRNA. b, c Representative Western blots images and quantification to detect protein levels of bax and bcl-2 in rat aortic AFs. d The detection of cell apoptosis by flow cytometry array with quantification in rat aortic AFs. e Dihydroethidium staining to measure ROS generation in rat aortic AFs. GAPDH was used as an endogenous control. n = 3–4 for each group except for a where n = 6. **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with ATII or ATII + NC siRNA group. A.U. arbitrary units, R.E. relative expression, AFs adventitial fibroblasts, ATII angiotensin II, NC negative control, ELA elabela, ROS reactive oxygen species

Fig. 2

Effects of ELA on cell migration and inflammatory response in rat aortic AFs in response to ATII. a Representative wound-healing images and quantification at 0 and 24 h in rat aortic AFs. b Relative mRNA levels of IL-1β, IL-6, MCP-1, and TNF-α in rat aortic AFs. c, d Protein levels of ACE2 and FGF21 in rat aortic AFs pretreated with ELA and ELA siRNA by Western blot analysis. e, f The mRNA levels of FGF21 and ACE2 were elevated by ELA stimulation but were reduced by ELA knockdown, respectively. g, h The concentration of FGF21 in the cell culture medium was measured in the presence of ATII, ELA, and ELA siRNA by ELISA kit. GAPDH was used as an endogenous control. n = 3–6 for each group. *P < 0.05, **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with ATII or ATII + NC siRNA group. A.U. arbitrary units, R.E. relative expression, AFs adventitial fibroblasts, ATII angiotensin II, NC negative control, ELA elabela, FGF21 fibroblast growth factor 21, ACE2 angiotensin-converting enzyme 2, IL-1β interleukin-1β, IL-6 interleukin-6, TNF-α tumor necrosis factor-α, MCP-1, monocyte chemoattractant protein-1

Fig. 3

FGF21 knockdown prevented the protective effects of ELA on ATII-mediated promtion of  apoptosis, inflammation, oxidative stress, and cell migration in rat aortic AFs. a Flow cytometry to detect cell apoptosis in rat aortic AFs in the presence of ATII, ELA, and FGF21 siRNA. b Representative Western blots images and qualification to measure the protein levels of bax and bcl-2 in rat aortic AFs. c Relative mRNA levels of IL-1β, IL-6, MCP-1, and TNF-α in rat aortic AFs by RT-PCR. d, e The level of ROS and cell migration was detected in rat aortic AFs by dihydroethidium staining and wound healing analysis, respectively. GAPDH was used as an endogenous control. n = 3–4 for each group except for c where n = 5–6. ##P < 0.01 compared with the ATII group. $P < 0.05, $$P < 0.01 compared with ATII + ELA + NC siRNA group; A.U. arbitrary units, R.E. relative expression, AFs adventitial fibroblasts, ATII angiotensin II, NC, negative control, ELA elabela; IL-1β, interleukin-1β, IL-6, interleukin-6, TNF-α tumor necrosis factor-α, MCP-1, monocyte chemoattractant protein-1, ROS, reactive oxygen species

Fig. 4

Regulatory roles of FGF21 in cell apoptosis, oxidative stress, cell migration, and inflammatory response in rat aortic AFs. a Flow cytometry to detect cell apoptosis in rat aortic AFs. b, c Western blot analysis to examine the protein levels of bax and bcl-2. d, e ATII-mediated the augmentation of oxidative stress and cell migration in rat aortic AFs were accelerated by FGF21 siRNA but was rescued by FGF21 stimulation. f The relative mRNA levels of proinflammatory cytokines in rat aortic AFs by RT-PCR. GAPDH was used as an endogenous control. n = 3–4 for each group except for f where n = 5. **P < 0.01 compared with the control group; #P < 0.05, ##P < 0.01 compared with ATII or ATII + NC siRNA group. A.U. arbitrary units, R.E. relative expression, AFs adventitial fibroblasts, ATII angiotensin II, NC negative control, FGF21 fibroblast growth factor 21, IL-1β interleukin-1β, IL-6 interleukin-6, TNF-α tumor necrosis factor-α, MCP-1 monocyte chemoattractant protein-1

Fig. 5

Protective roles of FGF21 in ATII-induced promotion of apoptosis, inflammatory response, oxidative stress, and cell migration were mediated by ACE2 signaling in rat aortic AFs. a The protein levels of ACE2 in the presence of ATII and ACE2 knockdown with siRNA. b-d Protein and mRNA levels of ACE2 in rat aortic AFs in response to ATII, FGF21, and FGF21 siRNA, respectively. e, f Cell apoptosis and apoptosis-associated proteins were detected by flow cytometry array and Western blots after exposure to ATII, FGF21, and ACE2 siRNA, respectively. g The expression of proinflammatory cytokines in rat aortic AFs by RT-PCR. h, i Dihydroethidium staining and wound healing assay to evaluate oxidative stress and cell migration in rat aortic AFs. GAPDH was used as an endogenous control. n = 3–4 for each group except for d and g where n = 5–6. *P < 0.05, **P < 0.01 compared with the control group. #P < 0.05, ##P < 0.01 compared with ATII or ATII + NC siRNA group. &P < 0.05, &&P < 0.01 compared with ATII + FGF21 + NC siRNA group; A.U. arbitrary units, R.E. relative expression, AFs adventitial fibroblasts, ATII angiotensin II, NC negative control, FGF21 fibroblast growth factor 21, ACE2 angiotensin-converting enzyme 2, IL-1β interleukin-1β, IL-6 interleukin-6, TNF-α tumor necrosis factor-α, MCP-1 monocyte chemoattractant protein-1

Fig. 6

Schematic overview of the crucial roles of Elabela in rat aortic adventitial fibroblasts. Elabela serves as a negative regulator of ATII-mediated the promotion of apoptosis, inflammatory injury, oxidative stress, and cell migration in rat aortic AFs by the activation of the FGF21–ACE2 signaling. ELA Elabela, FGF21 fibroblast growth factor 21, ACE2 angiotensin-converting enzyme 2, APJ G-protein coupled receptor, FGFR fibroblast growth factor receptor, ROS reactive oxygen species, IL-1β interleukin-1β, IL-6 interleukin-6, TNF-α tumor necrosis factor-α, MCP-1 monocyte chemoattractant protein-1