4-1. Ang II-mediated cardiac ACE2 upregulation in human cardiofibroblast
The transcript expression of human ACE2 was examined in the Ang II−treated HCF cells. The expression of ACE2 mRNA was markedly elevated (P < 0.01) in a
concentration-dependent manner in the HCF cells treated with Ang II (Figure 4-1A).
Moreover, the HCF cells stimulated with 1 μM of Ang II resulted in time-dependent increase in ACE2 mRNA over 24 h (Figure 4-1B). The HCF cells were pretreated with an AT1R inhibitor, valsartan, and then stimulated with Ang II to check the role of AT1R in the ACE2 upregulation. Ang II−induced effect on the upregulation of cardiac ACE2 expression was abolished when the HCF cells were pretreated with valsartan. The result confirms that AT1R signaling transduction pathway is involved in the cardiac ACE2 upregulation by Ang II
stimulation (Figure 4-1C).
Figure 4-1. The mRNA expression of human ACE2 in the HCF cells treated with Ang II.
The effect of Ang II in the regulation of ACE2 expression was determined in both
dose-dependent (A) and time-dependent manner (B). (C) Ang II-mediated ACE2 expression was abolished by an AT1R blocker, valsartan (Val). The results for each experiment were normalized to the density of GAPDH PCR product. The relative transcript expression of ACE2 was calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D. * and ** indicate P < 0.05 and P < 0.01 as compared with the control group, respectively. ‡ indicates P < 0.01 as compared with the Ang II treated only.
B.
C.
A.
4-2. Human AT1R and AT2R could be markedly increased after Ang II stimulation
The effect of Ang II in the regulation of human AT1R and AT2R were determined in time-dependent manner. The transcript expression of AT1R was markedly increased in time-dependent over 72 h. This result shows that the Ang II−mediated ACE2 mRNA
expression could be strengthened by even more AT1R (Figure 4-2A). It is to say that AT2R is thought to counteract the signals transmitted by the AT1R, eliciting vasodilatation,
inhibition of proliferation, NO production and apoptosis. The AT2R may play a homeostatic role in the regulation of blood pressure in animal models of hypertension. The transcript expression of AT2R was examined by Ang II treatment in time-dependent manner. The AT2R mRNA expression was upregulated over 48 h. This data suggest that AT2R
expression was also increased by Ang II stimulation. According to the parallel effect of Ang II−mediated AT1R and AT2R expression, we suggest that the ratio of AT1R/AT2R could be considerably regard as the critical indicator of Ang II-induced heart dysfunction (Figure 4-2B).
Figure 4-2. The mRNA expression of human AT1R and AT2R in the HCF cells treated with Ang II. The effect of Ang II in the regulation of AT1R (A) and AT2R (B) expression was determined in time-dependent manner. The results for each experiment were normalized to the density of GAPDH control. The relative transcript expression of AT1R was calculated according to the values of control group as 100%. The relative transcript expression of AT2R was calculated according to the fold change relative to control group as 1.0 fold.
Histograms of all values are expressed as the mean ± S.D. * and ** indicate P < 0.05 and P
< 0.01 as compared with the control group, respectively.
B.
A.
4-3. ERK
−MAPK cascade is involved in Ang II-mediated upregulation of cardiac ACE2
The effect of Ang II-induced ACE2 expression in the HCF cells was further confirmed by the experiments of blocking the downstream signaling of AT1R, ERK−MAPK cascade.
The HCF cells were pretreated with a MEK inhibitor, PD98059, and then treated with Ang II.
The result shows that the mRNA expression of ACE2 increased by AT1R-dependent effect could be abolished by the PD98059 pretreatment (Figure 4-3A). Hence, the signal molecules within ERK-MAPK pathway were explored by Western blot to analyze whether ERK-MAPK cascade is involved in Ang II−mediated upregulation of cardiac ACE2. The results indicate that an upregulated ACE2 combined with increased phosphorylated MEK1/2 and activated ERK1/2 was found in the HCF cells after Ang II treatment, and all of
upregulation of ACE2, phosphorylated MEK1/2 and activated ERK1/2 were attenuated in the Ang II-treated HCF cells as the cells pretreated with PD98059 (Figure 4-3B).
Figure 4-3. Role of ERK-MAPK signaling of AT1R in the ACE2 regulation by Ang II. (A) The effect of Ang II on ACE2 gene expression was determined in the HCF cells pretreated with AT1R blocker (valsartan, Val) and MEK1/2 inhibitor (PD98059) to confirm the
AT1R-dependent effect. (B) ERK-MAPK signaling, including phosphorylated MEK1/2 and activated ERK1/2, was examined by Western blot to check the Ang II-mediated ACE2
expression. The results for each experiment were normalized to the density of GAPDH.
The relative expression of ACE2, phosphorylated MEK1/2 and activated ERK1/2 were calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D. * and ** indicate P < 0.05 and P < 0.01 as compared with the control group, respectively. † indicates P < 0.05 as compared with the Ang II treated only.
B.
A.
4-4. NADPH oxidase signaling pathway is concerned with Ang II-stimulated ACE2 upregulation
NADPH oxidase signaling pathway in Ang II−AT1R axis was also analyzed to verify the role in Ang II-stimulated ACE2 upregulation in the HCF cells. The experimental result shows that the HCF cells pretreated with DPI, a NADPH oxidase inhibitor, could abolish the increase of ACE2 mRNA stimulated by Ang II (Figure 4-4A). Furthermore, hydrogen peroxide was used as a source of reactive oxygen species (ROS) to test the role of ROS in the ACE2 regulation in HCF cells. The transcript expression of cardiac ACE2 was markedly increased in the HCF cells after hydrogen peroxide treatment (Figure 4-4B).
Figure 4-4. Role of NADPH oxidase in the regulation of ACE2 by Ang II. (A) The effect of Ang II in regulation of ACE2 expression was determined in HCF cells pretreated with
NADPH oxidase inhibitor (DPI) to confirm AT1R-dependent effect by RT-PCR. (B) The effect of reactive oxygen species (ROS) in regulation of ACE2 was examined in HCF cells treated with hydrogen peroxide. The results for each experiment were normalized to the density of the GAPDH. The relative transcript expression of ACE2 was calculated
according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D. * and ** indicate P < 0.05 and P < 0.01 as compared with the control group, respectively. † and ‡ indicate P < 0.05 and P < 0.01 as compared with the Ang II treated only, respectively.
A. B.
4-5. Mas receptor is involved in the effect of Ang 1-7-mediated upregulation of ACE2
The effect of Ang 1-7 on cardiac ACE2 regulation was studied in the HCF cells. Ang 1-7 treatment could significantly increase the transcript and protein expression of cardiac ACE2 (Figure 4-5). For evaluate whether Mas receptor is involved in the effect of Ang 1-7−mediated upregulation of ACE2, the HCF cells were pretreated with a Mas receptor blocker, A779, and then treated with Ang 1-7. Both the mRNA and protein level of ACE2 upregulated by Ang 1-7 were depressed by the A779 pretreatment, which indicated that Mas receptor is associated with Ang 1-7-induced ACE2 upregulation.
Figure 4-5. The regulation of ACE2 in HCF cells after Ang 1-7 treatment. The effect of Ang 1-7 on the regulation of ACE2 expression was examined by RT-PCR (A) and Western blot (B). Ang 1-7 induced ACE2 was further confirmed by the cardiac cells pretreated with Mas receptor blocker, A779. The results for each experiment were normalized to the
density of the GAPDH. The relative transcript and protein expression of ACE2 was calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D. * and ** indicate P < 0.05 and P < 0.01 as compared with the control group, respectively. † and ‡ indicate P < 0.05 and P < 0.01 as compared with the Ang II treated only, respectively.
A. B.
4-6. ERK-MAPK cascade and NADPH oxidase signaling pathway were involved in the Ang 1-7 – ACE2 axis
The effect of Ang 1-7-induced ACE2 expression in the HCF cells was further confirmed by the experiments of blocking the downstream signaling of ERK−MAPK cascade and NADPH oxidase signaling pathway. The experimental result shows that the HCF cells pretreated with PD98059 and DPI could abolish the increase of ACE2 protein stimulated by Ang 1-7, respectively (Figure 4-6A). The results also indicate that an upregulated ACE2 combined with increased activated ERK1/2 was found in the HCF cells after Ang 1-7
treatment, and all of upregulation of ACE2 and activated ERK1/2 were attenuated in the Ang 1-7 treated HCF cells as the cells pretreated with PD98059 and DPI, respectively (Figure 4-6B).
Figure 4-6. Role of ERK-MAPK signaling of Mas receptor and NADPH oxidase in the ACE2 regulation by Ang 1-7. (A) The effect of Ang 1-7 on ACE2 protein expression was determined by Western blot in the HCF cells pretreated with Mas receptor blocker (A779), MEK1/2 inhibitor (PD98059) and NADPH oxidase inhibitor (DPI) to confirm the
Mas-dependent effect. (B) ERK-MAPK signaling, including phosphorylated MEK1/2 and activated ERK1/2, was examined by Western blot to check the Ang 1-7−mediated ACE2 expression. The results for each experiment were normalized to the density of GAPDH.
The relative expression of ACE2 was calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D. * and ** indicate P <
0.05 and P < 0.01 as compared with the control group, respectively. † indicates P < 0.05 as compared with the Ang 1-7 treated only.
B.
A.
4-7. ACE2 upregulation stimulated by Ang 1-7 might be independent to Ang II−AT1R pathway
The effect of Ang 1-7 on the regulation of AT1R, Ang II receptor, in the HCF cells was examined. The result shows that Ang 1-7 treatment was insignificantly influence the AT1R expression in HCF cells at both of the transcriptional and translational level (Figure 4-7).
To test the existence of Ang 1-7−AT1R pathway, HCF cells were pretreated with AT1R blocker (Valsartan) to confirm the AT1R-dependent ACE2 protein regulation. The result shows that HCF cells pretreating with Valsartan has no marked effect on Ang 1-7 induced ACE2 upregulation. The experimental results suggest the ACE2 upregulation stimulated by Ang 1-7 might be independent to Ang II−AT1R pathway (Figure 4-8).
Figure 4-7. The regulation of angiotensin II type I receptors in the HCF cells treated with Ang 1-7. The effect of Ang 1-7 on the regulation of AT1R gene (A) and protein (B) expression were determined in the HCF cells pretreated with A779 to confirm Mas
receptor-dependent effect. The results for each experiment were normalized to the density of the GAPDH. The relative expression of AT1R was calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D.
A. B.
Figure 4-8. Role of the possibility of AT1R-dependent effect in the ACE2 regulation by Ang 1-7. The effect of Ang 1-7 on ACE2 protein expression was determined by Western blot in the HCF cells pretreated with AT1R blocker (Val) to confirm the AT1R-dependent effect.
The results for each experiment were normalized to the density of GAPDH. The relative expression of ACE2 was calculated according to the values of control group as 100%.
Histograms of all values are expressed as the mean ± S.D. * indicate P < 0.05 as compared with the control group.
4-8. The interference of each specific inhibitor or blocker was ruled out
To confirm the specific regulated mechanisms involved in the modulation of ACE2 and to exclude the effect of inhibitor treatment alone, the influence of each inhibitor on ACE2 regulation was determined by Western blot in the HCF cells pretreated with each signaling specific inhibitor (Valsartan, A779, PD98059 and DPI) to check the effect of each inhibitor on ACE2 regulation (Figure 4-9). The experimental results suggest that there is no markedly effect by inhibitor treatment alone. Thus, we could exclude the possibility of the
interference of each specific inhibitor.
Figure 4-9. The influence of each signaling specific inhibitor on ACE2 regulation. The effect of specific inhibitor on ACE2 protein expression was determined by Western blot in the HCF cells pretreated with each signaling specific inhibitor (Val, A779, PD98059 and DPI) to check the effect of inhibitors on ACE2 regulation. The results for each experiment were normalized to the density of GAPDH. The relative expression of ACE2 was calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D.
4-9. ACE2 was major represented at the peripheral of cell membrane with both Ang II and Ang 1-7 treatment
Immunofluorescence localization and regulation of the ACE2 and AT1R in cardiovascular-related areas could provide still more clear knowledge for exploring the biological modulation of RAS. We evaluated the localization of AT1R and ACE2 using immunofluorescence in the HCF cells and further elucidating the modulation of Ang II and Ang 1-7 on these two molecules. The localization of ACE2 was major represented at the peripheral of cell membrane and partially distributed over the inner nucleus. The result shows that an abundant labeling of ACE2 was found in the HCF cells with both Ang II and Ang 1-7 treatment. The effect of Ang II−, Ang 1-7−mediated ACE2 expression were further abolished by specific antagonist, Valsartan and A779, respectively.
4-10. AT1R representing at the boundary of cell membrane was increased by Ang II treatment but not Ang 1-7
The localization of AT1R was approximately represented at the boundary of cell
membrane. The experimental result shows that intense AT1R-positive immunofluorescence was found in the cells treated with Ang II but was no apparent effect by Ang 1-7 treatment.
The images in Figure 4-10, 4-11 provide further validation for Ang II− and Ang 1-7−
mediated ACE2 expression and an actual presentation of AT1R and ACE2 localization in HCF cells. The quantification of the fluorescence expression of ACE2 and AT1R in HCF cells was calculated in Figure 4-12.
Figure 4-10. Localization and regulation of ACE2 and AT1R in HCF cells treated with Ang II.
HCF cells grown on cover slips were treated with Ang II for 24 h. The cells after treatment were washed, then fixed and double-immunostained for ACE2 and AT1R. Colocalization appears as yellow after merging the images of Alexa 488-tagged ACE2 (green) and Alexa 594-tagged AT1R (red). The nuclear marker, DAPI, is shown in blue. Micrographs of cross sections were taken at a magnification of 600x.
Figure 4-11. Localization and regulation of ACE2 and AT1R in HCF cells treated with Ang 1-7. HCF cells grown on cover slips were treated with Ang 1-7 for 24 h. The cells after treatment were washed, then fixed and double-immunostained for ACE2 and AT1R.
Colocalization appears as yellow after merging the images of Alexa 488-tagged ACE2 (green) and Alexa 594-tagged AT1R (red). The nuclear marker, DAPI, is shown in blue.
Micrographs of cross sections were taken at a magnification of 600x.
Figure 4-12. Quantification of the fluorescence expression of ACE2 and AT1R in HCF cells treated with Ang II and Ang 1-7. The fluorescence images were observed under Confocal Microscope and quantified by imaging software, Image J. (A) The effect of Ang II on the regulation of ACE2 and AT1R protein expression were determined in the HCF cells
pretreated with Val. (B) The effect of Ang 1-7 on the regulation of ACE2 and AT1R protein expression were determined in the HCF cells pretreated with A779. The density of the fluorescence expression of ACE2 and AT1R were calculated according to the area of DIC view. The relative expression of ACE2 and AT1R were calculated according to the values of control group as 100%. Histograms of all values are expressed as the mean ± S.D. * and ** indicate P < 0.05 and P < 0.01 as compared with the control group, respectively. † and ‡ indicate P < 0.05 and P < 0.01 as compared with the Ang II or Ang 1-7 treated only
B.
A.
4-11. Deletion mutation of ACE2 promoter and figure out the intense promoter activity with Ang II and Ang 1-7 treatment
Serial truncated human ACE2 promoter (Table 3-3) was introduced into luciferase-based reporter vector, pGL3-basic, to rough out which region of constructed promoter might contain critical regulatory activity of ACE2 (Figure 3-1). The analyzed data showed that human cardiac ACE2 promoter activity was markedly upregulated with transfection of pGL3
(-516/+20) constructs (Figure 4-13). The pGL3 (-516/+20) constructs was further incubated with Ang II and Ang 1-7 for 24 h to verify the role in Ang II− and Ang 1-7−mediated ACE2 promoter activity. The result showed that human cardiac ACE2 promoter activity was significantly upregulated with Ang II stimulation. Ang II-induced ACE2 promoter activity could be abolished when the HCF cells pretreated with Valsartan. This result indicated that ACE2 promoter activity was parallel to the transcriptional and translational expression of ACE2. In order to confirm whether the Ang 1-7 regulated ACE2 promoter activity was the same as the data we have shown in transcript and protein expression of cardiac ACE2, HCF cells were treated with Ang 1-7 for 24 h. The analyzed data showed that Ang 1-7 could remarkably increase the human cardiac ACE2 promoter activity, but this upregulation has no noticeably effect by A779 pretreatment (Figure 4-14). The upstream region of the ACE2 gene and specific promoter binding site were shown in Figure 4-15.
Figure 4-13. Deletion mutation analysis of the ace2 promoter region in the HCF cells.
Fragments of the 5'-flanking regions of the human ACE2 gene were fused to the firefly luciferase cDNA in the vector pGL3-basic. The position of the promoter fragments relative to transcription start site (+1) is indicated. HCF cells were transfected with the indicated reporter gene constructs. Cells were lysed 24 h after transfection, and luciferase activities were determined from duplicate wells. Five different constructs of pGL3-ACE2 plasmids are described in Table 3-3. Data are presented as relative fold changes of luciferase activity to the pGL3-basic construct. The diagram integrates results of three independent
experiments. Histograms of all values are expressed as the mean ± S.D.
Figure 4-14. The regulation of ACE2 promoter activity in the HCF cells treated with Ang II and Ang 1-7. Fragments of the 5'-flanking regions of the human ACE2 gene were fused to the firefly luciferase cDNA in the vector pGL3-basic. HCF cells were transfected with the reporter gene constructs including the ace2 promoter region of (-516/+20). The effect of Ang II and Ang 1-7 on the regulation of ACE2 promoter activity was determined in the HCF cells pretreated with Valsartan and A779 to confirm AT1R and Mas receptor-dependent effect, respectively. Cells were lysed 24 h after transfection, and luciferase activities were
determined from duplicate wells. Data are presented as relative fold changes of luciferase activity to the pGL3-basic construct. The diagram integrates results of three independent experiments. Histograms of all values are expressed as the mean ± S.D.
Figure 4-15. The upstream region of the ACE2 gene. The transcription start site (TSS) is marked by a downward arrow and is numbered as +1.
The consensus sequence for putative NF-kB transcription factor binding sites is underlined. The promoter region is defined according to the position relative to the transcription start site (+1) in ACE2 mRNA sequence (GenBank No. AF_291820)