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Local Augmented Angiotensinogen Secreted from Apoptotic Vascular Endothelial Cells Is a Vital Mediator of Vascular Remodelling.

Wu SJ, Soulez M, Yang YH, Chu CS, Shih SC, Hébert MJ, Kuo MC, Hsieh YJ - PLoS ONE (2015)

Bottom Line: After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT) was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs.Increases in the expression of AGT protein and angiotensin II (Ang II) were found in a serum-free medium conditioned by HUVECs (SSC).In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Vascular remodelling is a critical vasculopathy found in atheromatous diseases and allograft failures. The local renin angiotensin system (RAS) has been implicated in vascular remodelling. However, the mechanisms by which the augmented local RAS is associated with the initial event of endothelial cell apoptosis in injured vasculature remain undefined. We induced the apoptosis of human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) through serum starvation (SS). After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT) was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs. In addition, the expression of angiotensin-converting enzyme (ACE) mRNA was increased in VSMCs but decreased to 50% in HUVECs during the same apoptotic process. Increases in the expression of AGT protein and angiotensin II (Ang II) were found in a serum-free medium conditioned by HUVECs (SSC). The increased Ang II was suppressed using lisinopril (an ACE inhibitor) treatment. Moreover, the activation of ERK1/2 induced by the SSC in VSMCs was also suppressed by losartan. In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

No MeSH data available.


Related in: MedlinePlus

Detection of AGT and Ang II in a conditioned medium.(A) Western blot analyses of AGT expression in an SS medium with different incubation times (0 minutes, 30 minutes, 1 h, 6 h). CE, cell extract from HUVECs. PL, plasma from blood sample. M, ladder marker. Upper panel shows rapid (5 minutes) staining of protein bands on a PVDF membrane with the Ponceau S stain. (B) The Ang II concentration detected using an ELISA assay was highly increased in the SS medium (conditioned for 4 h) compared with that in the ECs conditioned in the serum-free medium for 1 minute (***P < 0.001). Conditioning ECs with lisinopril in an SS medium, SS+Lis, suppressed the production of Ang II (***P < 0.001), but suppression was not found in SS+Ben. ns, no significance. Data are expressed as the mean ± SE and are representative of 3 experiments. (C) Western blot analyses of phosphorylated ERK1/2 expressions in VSMCs exposed to N, SS, SSC, and ACEi-SSC (SS+Lis) for 1 h. The activation of phospho-ERK1/2 was suppressed in the ACEi-SSC group. (D) The activation of phospho-ERK1/2 in SSC was suppressed by 10 μM losartan.
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pone.0132583.g005: Detection of AGT and Ang II in a conditioned medium.(A) Western blot analyses of AGT expression in an SS medium with different incubation times (0 minutes, 30 minutes, 1 h, 6 h). CE, cell extract from HUVECs. PL, plasma from blood sample. M, ladder marker. Upper panel shows rapid (5 minutes) staining of protein bands on a PVDF membrane with the Ponceau S stain. (B) The Ang II concentration detected using an ELISA assay was highly increased in the SS medium (conditioned for 4 h) compared with that in the ECs conditioned in the serum-free medium for 1 minute (***P < 0.001). Conditioning ECs with lisinopril in an SS medium, SS+Lis, suppressed the production of Ang II (***P < 0.001), but suppression was not found in SS+Ben. ns, no significance. Data are expressed as the mean ± SE and are representative of 3 experiments. (C) Western blot analyses of phosphorylated ERK1/2 expressions in VSMCs exposed to N, SS, SSC, and ACEi-SSC (SS+Lis) for 1 h. The activation of phospho-ERK1/2 was suppressed in the ACEi-SSC group. (D) The activation of phospho-ERK1/2 in SSC was suppressed by 10 μM losartan.

Mentions: Because of the high expression of AGT mRNA in starved HUVECs, we determined whether AGT protein was secreted into the SSC medium by using an immunoblotting assay. The AGT protein was detected in the serum-free medium when HUVECs were incubated with SS for 30 minutes; however the protein was not found with the original SS (at the 0-minute line) (Fig 5C). Rode plasma AGT is a heterogeneous protein that migrated at approximately 62 kDa on SDS-PAGE, as previously reported [18]. Moreover, we further assessed whether Ang II was produced in SSC by using a commercial ELISA kit. In a parallel experiment, another 2 ACEi-SSC media, SS+Lis and SS+Ben, were examined. We found that Ang II was significantly detected in SSC compared with SS (P < 0.001, Fig 5B), and the production of Ang II in SSC was significantly inhibited in SS+Lis (P < 0.001). However, the inhibition of Ang II was not observed in SS+Ben.


Local Augmented Angiotensinogen Secreted from Apoptotic Vascular Endothelial Cells Is a Vital Mediator of Vascular Remodelling.

Wu SJ, Soulez M, Yang YH, Chu CS, Shih SC, Hébert MJ, Kuo MC, Hsieh YJ - PLoS ONE (2015)

Detection of AGT and Ang II in a conditioned medium.(A) Western blot analyses of AGT expression in an SS medium with different incubation times (0 minutes, 30 minutes, 1 h, 6 h). CE, cell extract from HUVECs. PL, plasma from blood sample. M, ladder marker. Upper panel shows rapid (5 minutes) staining of protein bands on a PVDF membrane with the Ponceau S stain. (B) The Ang II concentration detected using an ELISA assay was highly increased in the SS medium (conditioned for 4 h) compared with that in the ECs conditioned in the serum-free medium for 1 minute (***P < 0.001). Conditioning ECs with lisinopril in an SS medium, SS+Lis, suppressed the production of Ang II (***P < 0.001), but suppression was not found in SS+Ben. ns, no significance. Data are expressed as the mean ± SE and are representative of 3 experiments. (C) Western blot analyses of phosphorylated ERK1/2 expressions in VSMCs exposed to N, SS, SSC, and ACEi-SSC (SS+Lis) for 1 h. The activation of phospho-ERK1/2 was suppressed in the ACEi-SSC group. (D) The activation of phospho-ERK1/2 in SSC was suppressed by 10 μM losartan.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4492977&req=5

pone.0132583.g005: Detection of AGT and Ang II in a conditioned medium.(A) Western blot analyses of AGT expression in an SS medium with different incubation times (0 minutes, 30 minutes, 1 h, 6 h). CE, cell extract from HUVECs. PL, plasma from blood sample. M, ladder marker. Upper panel shows rapid (5 minutes) staining of protein bands on a PVDF membrane with the Ponceau S stain. (B) The Ang II concentration detected using an ELISA assay was highly increased in the SS medium (conditioned for 4 h) compared with that in the ECs conditioned in the serum-free medium for 1 minute (***P < 0.001). Conditioning ECs with lisinopril in an SS medium, SS+Lis, suppressed the production of Ang II (***P < 0.001), but suppression was not found in SS+Ben. ns, no significance. Data are expressed as the mean ± SE and are representative of 3 experiments. (C) Western blot analyses of phosphorylated ERK1/2 expressions in VSMCs exposed to N, SS, SSC, and ACEi-SSC (SS+Lis) for 1 h. The activation of phospho-ERK1/2 was suppressed in the ACEi-SSC group. (D) The activation of phospho-ERK1/2 in SSC was suppressed by 10 μM losartan.
Mentions: Because of the high expression of AGT mRNA in starved HUVECs, we determined whether AGT protein was secreted into the SSC medium by using an immunoblotting assay. The AGT protein was detected in the serum-free medium when HUVECs were incubated with SS for 30 minutes; however the protein was not found with the original SS (at the 0-minute line) (Fig 5C). Rode plasma AGT is a heterogeneous protein that migrated at approximately 62 kDa on SDS-PAGE, as previously reported [18]. Moreover, we further assessed whether Ang II was produced in SSC by using a commercial ELISA kit. In a parallel experiment, another 2 ACEi-SSC media, SS+Lis and SS+Ben, were examined. We found that Ang II was significantly detected in SSC compared with SS (P < 0.001, Fig 5B), and the production of Ang II in SSC was significantly inhibited in SS+Lis (P < 0.001). However, the inhibition of Ang II was not observed in SS+Ben.

Bottom Line: After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT) was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs.Increases in the expression of AGT protein and angiotensin II (Ang II) were found in a serum-free medium conditioned by HUVECs (SSC).In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.

ABSTRACT
Vascular remodelling is a critical vasculopathy found in atheromatous diseases and allograft failures. The local renin angiotensin system (RAS) has been implicated in vascular remodelling. However, the mechanisms by which the augmented local RAS is associated with the initial event of endothelial cell apoptosis in injured vasculature remain undefined. We induced the apoptosis of human umbilical vein endothelial cells (HUVECs) and vascular smooth muscle cells (VSMCs) through serum starvation (SS). After the cells were subjected to SS, we found that the mRNA expression of angiotensinogen (AGT) was increased by >3-fold in HUVECs and by approximately 2.5-fold in VSMCs. In addition, the expression of angiotensin-converting enzyme (ACE) mRNA was increased in VSMCs but decreased to 50% in HUVECs during the same apoptotic process. Increases in the expression of AGT protein and angiotensin II (Ang II) were found in a serum-free medium conditioned by HUVECs (SSC). The increased Ang II was suppressed using lisinopril (an ACE inhibitor) treatment. Moreover, the activation of ERK1/2 induced by the SSC in VSMCs was also suppressed by losartan. In conclusion, we first demonstrated that the augmented AGT released from apoptotic endothelial cells acts as a vital progenitor of Ang II to accelerate vascular remodelling, and we suggest that blocking local augmented Ang II might be an effective strategy for restraining intimal hyperplasia.

No MeSH data available.


Related in: MedlinePlus