| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TRANSLATIONAL PHYSIOLOGY
1Division of Cardiac Surgery and 2Cardiology, 3Keenan Research Centre, Li Ka Shing Knowledge Institute, St. Michael's Hospital, Toronto, Ontario, Canada; 4Departments of Surgery, 5Medicine and 6Physiology, Division of Cardiology, 7University Health Network, 8R. Samuel McLaughlin Centre for Molecular and Regenerative Medicine, 9Heart and Stroke Richard Lewar Centre of Excellence, 10Interdepartmental Division of Critical Care, University of Toronto, Toronto, Ontario, Canada; 11Division of Vascular Surgery, College of Medicine and King Khalid University Hospital, King Saud University, Riyadh, Kingdom of Saudi Arabia; and 12IMBA, Institute for Molecular Biotechnology of the Austrian Academy of Sciences, Vienna, Austria
Submitted 29 March 2008 ; accepted in final form 11 July 2008
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
endothelium; atherogenesis; angiogenesis
The renin-angiotensin system (RAS) is a pivotal transducer of cardiovascular function and plays a causal role in the development and progression of endothelial dysfunction and atherosclerosis (7, 15, 17). RAS supports a series of complex enzymatic reactions that culminate in the generation of ANG II, the main proinflammatory effector of adverse cardiovascular phenotypes. Recently, a homologue of angiotensin converting enzyme (ACE), namely ACE2 has been identified, which serves as a negative regulator of the RAS (5, 6, 11). Unlike ACE, which processes ANG I to ANG II, ACE2 functions to promote the degradation of ANG II into the anti-inflammatory, vasodilator, and antitrophic heptapeptide, ANG-(1-7) (5, 6, 11). ACE2 plays a critical role in regulating ANG II production by ACE, either by stimulating an alternative pathway for ANG I degradation or by facilitating the degradation of ANG II into ANG-(1-7). Through counterbalancing the function of ACE, ACE2 may determine net ANG II bioavailability in tissues. We and others (3, 12, 18) have recently demonstrated that ACE2 expression was upregulated in failing hearts and that ACE2-deficient mice exhibited progressive impairment of heart contractility and renal disease. Importantly, this phenotype could be rescued by loss of ACE as evidenced in ACE/ACE2 double knockout (KO) mice, implicating a fundamental role of ACE2 in regulating ANG II bioactivity.
ACE2 is highly expressed in vascular endothelial cells (as opposed to vascular smooth muscle cells or cardiomyocytes; Refs. 3, 4, 6, 11), where it may potentially regulate endothelial cell responses to inflammation by limiting the effects of ACE-mediated ANG II production. Improving endothelial function may be a central mechanism through which ACE2 exerts cardiovascular and renal protection; however, the role of ACE2 in endothelial homeostasis remains unknown. Using gain and loss of function strategies respectively, we systematically evaluated the role of ACE2 in endothelial regulation. Furthermore, since atherosclerosis represents a clinically relevant endpoint of progressive endothelial dysfunction we examined the potential of ACE2 to limit experimental atherosclerosis.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Mice. Animal procedures were in accordance with the guidelines of the Canadian Council on Animal Care and approved by the Institutional Animal Care Committee. Male ACE2-KO mice have been described previously (3), and male wild-type (WT) littermates acted as controls (10–12 wk old). Baseline measurements of systolic blood pressure were similar between male ACE2-KO mice (131 ± 4 mmHg) and male WT littermate controls (136 ± 4 mmHg).
Cells. Endothelial cells were isolated from the lungs of 6-wk-old ACE2-KO mice and WT littermates as described previously (8). Human umbilical vein endothelial cells (HUVEC; Clonetics) and THP-1 cells (ATCC) were grown respectively in MCDB-131 complete medium and in RPMI 1640 (ATCC), both supplemented with 10% FBS.
Functional studies. Mice were killed by cervical dislocation, and 2-mm segments of thoracic aortas were mounted in a wire myograph for measurement of isometric tension. Vessel segments were equilibrated in aerated Krebs-Henseleit solution for 1 h at 37°C under a resting tension of 1,000 mg. Aortic segments were preconstricted with 1 µM phenylephrine and relaxation-response curves to acetycholine or SNP were generated. Vasorelaxation was calculated as a percentage of the phenylephrine-induced maximal response.
ACE2 mRNA overexpression and silencing. For overexpression experiments, HUVEC culture medium was replaced with that containing AdACE2- or AdGFP (100 multiplicity of infection) for 6 h. Silencing experiments were performed in the presence of 25 nM small-interfering RNA (siRNA) ACE2 (Ambion) in OptiMEM (Invitrogen) using siSPORT NeoFX (Ambion). Cell lysates were collected after 48 h.
Studies with ANG-(1-7) inhibition. AdACE2- and AdGFP-transfected HUVEC were incubated for 30 min with vehicle or the ANG-(1-7) inhibitor A779 (10 µM, Bachem) before being exposed to ANG II (0.1 µM) for 18 h.
RNA analyses. Real-time PCR was performed with the following primers: 5'-CATTGGAGCAAGTGTTGGATCTT-3' and 5'-GAGCTAATGCATGCCATTCTCA-3' for ACE2, 5'-GAGCGGCATCTACCTACTGG-3' and 5'-TGATGGTGCCTCCGATCT-3' for p22phox, 5'-CACCAGGGCTGCTTTTAACTCTGGTA-3' and 5'-CCTTGACGGTGCCATGGAATTGC-3' for GAPDH. Monocyte chemoattractant protein 1 (MCP-1) primers were obtained from SuperArray. mRNA expression was analyzed using the method of Pfaffl (13). The relative abundance of RNA expression in untreated control cells was expressed as 100% for comparative purposes.
Protein analyses. HUVEC protein lysates (30 µg) were resolved on 10% SDS-PAGE and electrotransferred onto nitrocellulose. Blots were blocked in 5% milk-TBS-Tween before incubation with mouse anti-ACE2 (R&D Systems), rabbit anti-p22phox (Santa Cruz Biotechnology), or mouse anti-actin (Chemicon International) antibodies. Immunoblots were probed with secondary antibodies and visualized by enhanced chemiluminescence (Amersham). Band intensity was quantified by densitometry.
ACE2 activity. ACE2 enzymatic activity in the culture medium was assayed using 7-methoxycoumarin-Tyr-Val-Ala-Asp-Ala-Pro-Lys-(2,4-dinitrophenyl)-OH (R&D Systems).
Tube formation assay. Angiogenic activities were monitored in AdACE2-transfected HUVEC, siRNA ACE2-transfected HUVEC and murine lung endothelial cells. Where required, endothelial cells were treated with 1 µM losartan for AT1 receptor blockade. Cells (5 x 105) were cultured in Matrigel basement membrane matrix (BD Bioscience) coated 12-well plates. Cumulative tube length was quantified after 18 h with a NIH Image analysis software (ImageJ) and evaluated in 5 random fields.
Migration assay. HUVEC treated with AdACE2 or siRNA ACE2 were placed in the upper chamber of a modified Boyden chamber (5 x 104 cells per chamber; pore size = 8 µm; BD Bioscience). Endothelial cell migration in response to 24 h of VEGF (50 ng/ml) exposure was assessed in three random fields after staining with a Diff-Quik Stain kit (Dade Behring).
Proliferation. HUVEC were seeded at a density of 1 x 104 cells/well in 96-well plates, treated with Ad-ACE2 or ACE2 siRNA and incubated for 24 h with bromodeoxyuridine (BrdU) that was added to a final concentration of 10 µM. BrdU incorporation into newly synthesized cellular DNA acting as a surrogate for cell proliferation was determined with an enzyme-linked immunosorbent assay (Roche Diagnostics).
Static adhesion.
AdACE2-transfected HUVEC and murine lung endothelial cells were plated at a density of 2 x 104 cells/well, stimulated for 6 h with TNF-
(10 ng/ml) and overlaid with PKH26-labeled THP-1 monocytes (5 x 105) for 30 min. Non-adherent THP-1 monocytes were washed off and the percentage of adherent monocytes correlated to the fluorescence intensity measured with a 544 nm/590 nm filter set.
Reactive oxygen species formation. After various treatments, HUVEC were incubated for 45 min with 10 µM 5(and 6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, Molecular Probes). Intra-cellular reactive oxygen species (ROS) generation was assessed by measuring fluorescence at excitation and emission wavelengths of 485 nm and 535 nm respectively.
Vascular cell adhesion molecule-1 secretion. Levels of soluble Vascular cell adhesion molecule-1 (VCAM-1) protein in HUVEC culture media were measured with an ELISA kit (Biosource).
In vivo matrigel plug assay. C57Bl/6 mice were injected with Matrigel HC (0.5 ml; BD Bioscience) complexed with PBS, VEGF (200 ng), AdGFP [2.5 x 108 plaque-forming unit (PFU)] or AdACE2 (2.6 x 108 PFU). Blood vessel infiltration was quantified after 7 days by analysis of Cy3-labeled smooth muscle actin-staining.
Atherosclerotic lesions. Apolipoprotein (apo) E-KO mice (12 wk old) were placed on the Western diet for 4 wk and injected via the tail vein with AdACE2 or AdGFP (1–1.1 x 109 PFU). Paraformaldehyde-fixed hearts and proximal aortas were excised for staining of aortic sinus sections (10 µm) with oil red O.
Immunohistochemistry. Macrophage infiltration was determined in the ascending aortas of adenovirus-treated apoE-KO mice by probing cryosections with a rat anti-mouse macrophage antibody (clone MOMA-2, Chemicon). Three sections from each mouse were examined.
Statistical analysis. Results are means ± SE for number of animals per samples with P < 0.05 representing significance. Where appropriate, means were compared by unpaired two-tailed Student's t-test. Differences among multiple means were evaluated by ANOVA.
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
|
|
-induced monocyte-endothelial adhesion whereas ACE2 silencing increased monocyte-endothelial interaction (Fig. 3D). ACE2 modulates ANG II-induced ROS production in endothelial cells. The major source of ROS in endothelial cells is NADPH oxidase. ANG II stimulates membrane-bound NADPH oxidase which in turn generates ROS within endothelial cells(14). ANG II-induced ROS generation, as assessed by dihydroethidium fluorescence were attenuated by ACE2, and this effect was attenuated by inhibition of ANG-(1-7) with A779 (Fig. 4A). We extend these observations further by providing data that the effects of ANG II to upregulate the expression of p22phox (protein and transcript) are attenuated by ACE2. These effects are also inhibited by A779 (Fig. 4, B and C). ANG II-induced increases in MCP-1 and VCAM-1 in endothelial cells are likewise attenuated by ACE2, and these effects are also blunted during coincubation with A779 (Fig. 4, D and E). These data suggest that ACE2, in a ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis, via a mechanism that may involve attenuation of NADPHox-induced ROS production.
|
-induced monocyte-endothelial cell adhesion (Fig. 5), consistent with the observations in ACE2-silenced HUVEC.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
Using gain- and loss-of-function strategies respectively, we have carefully delineated the role of ACE2 in endothelial homeostasis, inflammation and atherosclerosis. We demonstrate that aortas from ACE2-deficient mice exhibit impaired endothelium-dependent vasodilation. Furthermore, silencing of the ACE2 transgene in isolated human endothelial cells results in decreased tube formation and migration, a phenotype that is also observed in endothelial cells isolated from ACE2-KO mice. Overexpression of ACE2 improves endothelial cell migration and tube formation. Endothelial cell dysfunction uncovers a proinflammatory milieu which promotes monocyte adhesion to endothelial cells, and an increase in cellular adhesion molecules, which in turn sets the stage for the development of fatty streaks and early atheroma. Therefore, we evaluated the role of ACE2 on endothelial-monocyte interaction. Endothelial overexpression of ACE2 inhibited TNF- induced monocyte adhesion, whereas silencing of ACE2 increased monocyte-endothelial adhesion. Furthermore, ANG II-induced increases in cellular adhesion molecules, VCAM-1, and MCP-1 were attenuated in ACE2 overexpressed endothelial cells. These data suggest that ACE2 plays a permissive role in limiting endothelial cell inflammation, both under inflammatory and ANG II stimulated conditions.
One of the important effectors of ANG II is the generation of ROS. We demonstrate that endothelial ANG II-induced ROS generation, is attenuated by ACE2. To confirm if this effect is mediated, in part, through ANG-(1-7), we utilized A779, an antagonist to the ANG-(1-7) receptor. The effects of ACE2 to limit ANG II-induced ROS generation were attenuated by A779 confirming that ACE2 functions largely through an ANG-(1-7)-dependent pathway. We extend these observations further by demonstrating that the effects of ANG II to upregulate the expression of p22phox (protein and transcript) are attenuated by ACE2, and that these effects are also inhibited by A779. ANG II-induced increases in MCP-1 and VCAM-1 in endothelial cells are likewise attenuated by ACE2, and these effects are also blunted during coincubation with A779. Therefore, we believe that ACE2, in a ANG-(1-7)-dependent fashion, functions to improve endothelial homeostasis, via a mechanism that may involve attenuation of NADPHox-induced ROS production.
One of the important functions of endothelial cells is promoting angiogenesis, and hence we investigated the effects of ACE2 on neovascularization capacity. ACE2 impregnated Matrigel demonstrated an elevated number of infiltrating vessels indicating that ACE2 may function in vivo to promote endothelial cell mediated neovascularization.
Atherosclerosis is the cardiovascular endpoint of progressive endothelial cell dysfunction, and hence we evaluated whether an ACE2-based strategy would limit atherosclerosis in apoE-KO mice. Four wk treatment of atherosclerosis prone mice with AdACE2 revealed a significant decrease in oil red-O-positive areas compared with that of mice injected with AdGFP. Additionally, in apoE-KO mice injected with AdACE2 macrophage infiltration was less prominent than in control mice aorta, consistent with the notion that that ACE2 may function to limit aberrant endothelial function, which culminate into reduced atherosclerosis.
It is important to mention that ANG-(1-7) has been demonstrated to inhibit endothelial tube formation, and endothelial proliferation (1, 10) whereas other studies have demonstrated that Ang1-7 inhibits vascular smooth muscle proliferation (16) and stimulates proliferation of endothelial progenitor cells (9). Although the effects of Ang1-7 on vascular biology are conflicting, our data set suggest that ACE2 may exert endothelial protection, in part via ANG-(1-7), however, they do not exclude the possibility that other non-ANG-(1-7) pathways may be involved in ACE2 mediated vasculoprotection.
Taken together, these data identify ACE2 as a novel target of endothelial cell function, and endothelial responses to injury. The endothelium is a key target of ACE2 activation, and serves to limit inflammation and ANG II-mediated vascular dysfunction. We postulate that via improving endothelial repair and regeneration, ACE2 plays a permissive role in reducing atherosclerosis. Pharmacological and cell based strategies aimed at increasing vascular ACE2 bioavailability may represent not only a new therapeutic avenue in the treatment of atherosclerotic cardiovascular disease, but as a target for various cardiometabolic, pulmonary, septic, and renal diseases where endothelial dysfunction is an etiological factor.
|
GRANTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
* Y. Pan and A. Quan contributed equally to this work. 
|
REFERENCES |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  C. T. Chan, F. Lovren, Y. Pan, and S. Verma Nocturnal haemodialysis is associated with improved vascular smooth muscle cell biology Nephrol. Dial. Transplant., September 17, 2009; (2009) gfp495v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 F. Lovren, Y. Pan, P. C. Shukla, A. Quan, H. Teoh, P. E. Szmitko, M. D. Peterson, M. Gupta, M. Al-Omran, and S. Verma Visfatin activates eNOS via Akt and MAP kinases and improves endothelial cell function and angiogenesis in vitro and in vivo: translational implications for atherosclerosis Am J Physiol Endocrinol Metab, June 1, 2009; 296(6): E1440 - E1449. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P < 0.05 vs. AdGFP; 
P < 0.05 vs. siRNA control. C: AdACE2 and siRNA ACE2 transfection respectively augment and diminish HUVEC migration. Data are means ± SE; n = 3–7; *P < 0.05 vs. control; 
