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Genetic Ace2 deficiency accentuates vascular inflammation and atherosclerosis in the ApoE knockout mouse.

Interview with Dr. Tikellis

Q: Dear Dr. Tikellis, you really aced a study published in Circulation Research on the first of October. It is on the other, often overlooked side of the renin-angiotensin system, namely the enzyme that degrades angiotensin II (Ang II), i.e. Ang II-converting enzyme (ACE2). We are so used to look at the Ang I-converting enzyme, would you mind pointing out to us the key effects of genetic ACE2 deficiency on the vascular wall that you observed in your study in combination and apart from ApoE deficiency including inflammation, adhesion of inflammatory cells, and atherosclerosis?     

A: It is well known that the RAS has an important role in atherosclerosis.  All the RAS components are present in the vessel wall and angoitensin II is a driving factor in plaque formation by promoting monocyte and endothelial cell activation.  For quite a while we have been trying to think of ways to decrease Ang II levels, mostly by reducing its synthesis by Ang II-converting enzyme (ACE). However, Ang II levels are also regulated and balanced by those enzymes that break Ang II down, the most important of which appears to be ACE2.

When ACE2 activity is deficient, tissue and circulating levels of Ang II increase. In addition, there is a fall in the level of Ang 1-7, the major product of ACE2 and important vasodilator and anti-inflammatory molecule.  The net effect means an increase in systolic blood pressure, cardiac hypertrophy, and in genetically predisposed mice, and increase in atherogenesis.

Q: ACE2 deficiency by itself, i.e. in the absence of any genetic susceptibility, did not lead to atherosclerosis despite elevated circulating and tissue levels of Ang II and induction of proinflammatory mediators – is this correct? Is this a reflection of the animal model, the follow-up time etc. or does it indicate that Ang II by itself is insufficient to induce atherosclerosis? 

A:  Yes, this is fundamental limitation of mouse research. Due to very efficient lipoprotein metabolism in mice, a pro-atherogenic phenotype is required for the development of an atherosclerotic plaque.  In this study we used the ApoE KO mouse, the model most widely employed to investigate experimental atherosclerosis, with plaque accumulation and morphology in this strain resembling human disease. This does not mean that Ang II cannot induce atherosclerosis on its own, rather that almost nothing can in mice, unless the substrate to form plaque (fat) is also present.

We specifically studied the effects of ACE2 in the absence of dyslipidemia to examine the pro-atherogenic mediators specifically associated with ACE2 deficiency, without the confounding effects of atherosclerosis, where changes associated with vascular disease could be interpreted as being epiphenomena rather than causative in the disease process. In this light we observed that ACE2 deficiency was associated with significant up-regulation of pro-inflammatory pathways even in the absence of atherosclerosis, albeit to a lesser extent, suggesting that the increased inflammation in ApoE/Ace2 double KO mice was not simply an epiphenomenon associated with enhanced plaque accumulation.

Q: On a more provocative note, could the latter finding of lack of development of atherosclerosis despite an increase expression of many proinflammatory mediators indicate that inflammation is not enough for atherosclerosis?    

A: Again, unless the substrate to form plaque (fat) is also present, the stimulus (Ang II, inflammation, etc) will not be able to cause significant plaque development, due to very efficient lipoprotein metabolism in mice. This is a very different case in humans, and particular Westernized humans in whom the lipid profile is ripe for atherogenesis.

Q: I believe you also did immunostaining of ACE2. Where did you observe the expression in the vascular wall in normal animals and how was this changed in ApoE knockout mice? Is it up- or downregulated in the atherosclerotic wall?

A:  The vascular expression of ACE2 is largely in the endothelial cell layer of the vessel.  However, activated macrophages, and smooth muscle cells also express ACE2, albeit at lower levels. The apoE KO mouse does have modestly lower expression than wild type controls.  We have previously shown that ACE2 expression in the endothelium is downregulated in diabetic mice.

Q:  Did you notice any co-localization of ACE2 with other peptides that you studied?

A:  In this study we did not stain for other peptides. ACE2 is usually co-localised with other components of the RAS including ACE and the AT1 receptor.

Q: Did you measure ACE2 activity? Some studies suggest that its activity is unregulated in atherosclerotic plaques, possibly as a compensatory mechanism. Is there a reverse feedback mechanism?

A:  Our studies looked at ACE2 deficient mice that completely lack ACE2 activity, meaning that it was not necessary in the present study. We have previously measured ACE2 activity in whole aortas and in the circulation and have found a reduction in ACE2 activity correlating with an increase in plaque burden.

Q: How did Perindopril influence these dynamics? Were there additional hemodynamic changes that you observed, specifically what about the blood pressure profile? 

A: Treatment with the ACE inhibitor, perindopril reduced atherosclerotic plaque area in the aorta of Ace2/ApoE double KO mice. This is consistent with the anti-atherosclerotic effects of RAS blockade observed in other models. However, the degree of protection was significantly less than that observed following treatment in ApoE KO mice, despite a comparable reduction in blood pressure and circulating Ang II levels. This suggests that our Ace2 KO mice had a degree of resistance to the effects of ACE inhibition, a phenomenon that may be highly relevant to the clinical situation.

Q: Next you studies macrophages isolated from wild-type mice and ACE2 knockout mice. What did you find?

A:  We isolated bone marrow macrophages from C57Bl6 and ACE2 KO mice.  Macrophages from ACE2 KO mice has increased expression of pro-inflammatory genes including VCAM-1, IL-6, TNF, MCP-1 and MMP9 when compared to C57Bl6 mice.  In addition, when these macrophages were activated with LPS they secreted higher levels of the inflammatory cytokines, TNF and IL-6 (measured in the media) when compared to TNF stimulated C57Bl6 macrophages. Taken together this data suggests that ACE2 deficiency leads to a pro-inflammatory phenotype, consistent with the known actions of Ang II excess on inflammatory cells.

Q: You also isolated endothelial cells from the aorta of these mice. Did you observe a proinflammatory switch in them as well?

A: Yes, we did. In our experiments, primary endothelial cells from Ace2 KO mice also expressed elevated gene mRNA levels for VCAM-1, IL-6 and MCP-1 when compared to endothelial cells isolated from C57Bl6 mice.  Again, activation of these cells, this time with TNF treatment further increased the expression levels of these genes in ACE2 deficient cells.  In addition, the ACE2 KO endothelial cells also secreted more soluble VCAM into the cell media when compared to endothelial cells from C57Bl6 mice and this was further increased by TNF treatment.

To back up these findings we also carried out flow chamber studies where labeled human leukocytes were circulated though C57Bl6 and Ace2 KO mouse aortas.  This functional study showed that when these labeled cells passed through an Ace2 KO mouse aorta more leukocytes adhered to the vessel wall, demonstrating the functional pro-inflammatory state of these Ace2 KO mice.

Q: Based on your observation, would you say that the effects are mainly due to reduced Ang II levels or generation of the degradation product Ang (1-7)? Are you planning further separate studies focussing on Ang (1-7)?   

A: We believe that ACE2 deficiency increases plaque accumulation both through increasing Ang II and reducing Ang 1-7. It is not appropriate to tease out single actions from a homeostatic pathway. It is very clear that Ang II on its own will cause atherosclerosis, and that Ang 1-7 will protect against it.

Q: How do these findings translate to the bedside? Shall we work on drugs enhancing ACE2 activity? Other than by expression, how is ACE2 activity regulated?

A:  RAS blockade is a major therapeutic strategy in the prevention and management of CVD. However, conventional RAS blockade is often encumbered by suboptimal efficacy and escape (ie significant levels of Ang II are still formed). In cancer therapy the best approaches appear to require multiple agents with broad and complementary coverage. The same is probably the case for RAS blockade. We anticipate that enhancing ACE2 activity will prove therapeutically important.  Some drugs have been designed to specifically activate ACE2 while recombinant ACE is also being tested.  It is quite reasonable to imagine treating a patient with an ACE inhibitor (or AT1R blocker) alongside a compound that enhances ACE2 activity to get a more complete blockade of the RAS.

Q: What are the next steps that are taken in the field of ACE2 research? What are the open questions that you see?

A: The renin angiotensin system is not an old fashioned linear pathway, but a complex homeostatic regulator composed of a number of different regulatory components and effector molecules that facilitate the dynamic control in both health and disease.  Our research has highlighted an important role for ACE2 deficiency in atherogenesis. It is now up to researchers to develop novel ways to redress this deficiency, which our work provides a potential model.



Q: Again, thank you so much for this interview and discussing this terrific study of yours. Hopefully, this will stimulate further research in this area.

A:    Thanks for your interest in our work.

The content of this article reflects the personal opinion of the author/s and is not necessarily the official position of the European Society of Cardiology.

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