In order to bring you the best possible user experience, this site uses Javascript. If you are seeing this message, it is likely that the Javascript option in your browser is disabled. For optimal viewing of this site, please ensure that Javascript is enabled for your browser.
Did you know that your browser is out of date? To get the best experience using our website we recommend that you upgrade to a newer version. Learn more.

We use cookies to optimise the design of this website and make continuous improvement. By continuing your visit, you consent to the use of cookies. Learn more

Circulating endothelial progenitor cells do not contribute to plaque endothelium in murine atherosclerosis

Basic Sciences, Pharmacology, Genomics and Cardiovascular Pathology


Q: Thank you, Mette, for discussing your extraordinary study that was published in Circulation in February 2010. Your study is extraordinary as it addressed or shall we say challenged the view that circulating endothelial progenitor cells contribute/replace plaque endothelium. You chose an extraordinarily elaborate approach to get the answer as well. Would you mind summarizing in a few words would you did and what you found?  

A: We studied the origin of plaque endothelial cells (ECs) in apolipoprotein E-deficient (apoE-/-) mice during atherogenesis and after mechanical plaque disruption with superimposed thrombosis. Either recipient mice, the bone marrow (BM) or the common carotid artery isograft expressed enhanced green fluorescence protein (eGFP) and, consequently, ECs over atherosclerotic plaques could easily be tracked and distinguished from recruited circulating cells. Also, the Y chromosome was used as an independent tracking marker in mismatch transplantations between female and male mice. Using these series of transplantation techniques, specific markers for cell type and origin, and high-resolution microscopy we found that ECs in both intact and healed disrupted plaques are replenished by local proliferation with no or extremely rare contributions from circulating endothelial progenitor cells.

Q: In essence, you looked at endothelial cell origin in atherosclerosis and after plaque disruption form two possible sources: bone marrow and blood. How exactly did you distinguish the two origins and which markers for EC did you use? 

A: To evaluate the potential contribution of endothelial progenitor cells (EPCs) of hematopoietic origin to plaque ECs we lethally irradiated apoE-/- mice and reconstituted them with eGFP+ BM cells. Thus, if there was a contribution from EPCs of hematopoietic origin, then ECs would be eGFP+. To confirm our findings that ECs are not regenerated by BM derived cells and to evaluate if there was a contribution from EPCs circulating in the blood, we then transplanted common carotid arterial segments from apoE-/- mice into eGFP+apoE-/- mice and induced atherosclerotic plaques within the grafts. Again, if there was a contribution from circulating EPCs then these cells would be eGFP+.

As EC marker, we used von Willebrand Factor (vWF) which is a specific EC marker when identified within nucleated cells, usually with a granular cytoplasmic pattern, by single-cell resolution microscopy. Although vWF is not detectable in all ECs in the mouse it is, however, robustly expressed in larger arteries and restricted to ECs. ECs, defined as nucleated cells with clear intracellular location of vWF, were then analyzed for co-localization of eGFP signal.

Q: In the first experiment, did you see a difference in the extent of atherosclerosis among the various segment of the aorta and the brachiocephalic trunk, the two time points (20 or 32 weeks) and any difference with regards to the contribution of bone marrow-derived stem cells to it?

A: We only analyzed atherosclerotic plaques in the aorta at 20 weeks of age but at 32 weeks of age, all mice had developed fibrofatty plaques in the aortic root, aortic arch, brachiocephalic trunk, abdominal aorta, and to a variable extent in the descending thoracic aorta. The extent of atherosclerosis was higher after 32 weeks than 20 weeks. It seemed as if the contribution of BM-derived cells (i.e. macrophages) was similar in the various segments.

Q: In the second experiment, you used a constrictive perivascular collar model in transplanted CCA segments. How representative is this for “natural” atherosclerosis     

A: An extensive number of observations support the assumption that the pathogenesis of collar-induced atherosclerosis is equivalent to that of spontaneously developing atherosclerosis. Lesions in the constrictive collar model develop immediately proximal to the collar, presumably elicited by low wall shear stress in this region, and are strictly dependent on the presence of hypercholesterolemia. Thus, lesion development in this model appears to depend on the two key etiologic factors known for spontaneous atherosclerosis. Furthermore, constrictive collar induced lesions are pathoanatomically reminiscent of spontaneously developing atherosclerosis with respect to the presence of macrophage foam cells, cholesterol crystals, fibrous caps, and necrotic cores.

Q: The positive control rate in the gender-mix component of the two experimental setups was 40-50%; intriguing question, but shouldn’t it be 100%?

A: 40-50% is in the range predicted from the thickness of the section and the nuclear size when analyzing for the Y chromosome. On average only about half of the nucleus volume is contained within the section. Only if the Y chromosome is located in this part, it can be detected.

Q: In the third and fourth experiment, how did you guide and verify that truly atherosclerotic plaques were targeted for disruption? Did you visualize by ultrasound? How did you confirm it was done the same way with the same procedural result in all animals?    

A: We verified that atherosclerotic plaques were targeted for disruption by histological examination. The mechanical plaque disruption procedure caused consistent plaque surface injury with loss of plaque endothelium and the formation of a platelet-rich thrombusin all of the mice killed after 30 minutes. Like superimposed thrombi in humans, the thrombi were platelet rich, demonstrating that they had formed under rapid blood flow. After four weeks, no residual luminal thrombi were observed.  The healed plaques were covered by an intact layer of ECs indicating that endothelial regeneration had occurred and focal and distinct accumulations of smooth muscle cells were seenthat resembled healed rupture sites in humans. 

Q: For the mechanical plaque disruption studies, you made the same observations though: endothelial cells are (re-) generated locally – is that correct? 

A: Yes, in consistence with the first experiments on ECs regeneration over atherosclerotic plaques we found that ECs after mechanical plaque disruption are regenerated from local proliferation of ECs.

A: Yes, in consistence with the first experiments on ECs regeneration over atherosclerotic plaques we found that ECs after mechanical plaque disruption are regenerated from local proliferation of ECs.

Q: The cells from which endothelial cells generate in atherosclerosis or regenerate after injury are then the local endothelial cells or do we have to consider other local cell populations?

A: Our study was not designed to investigate whether the migrating recipient cells were mature, proliferating ECs. We believe this to be a very likely mechanism, but cannot exclude the potential involvement of putative local stem cells in the arterial wall. However, as far as we know, there have been no studies claiming that local arterial stem cells can give rise to ECs.

Q: This leads up to the question if dysfunctional endothelial cells regenerate well? Isn’t there regeneration potential supposed to be lower as well? How does it compare do the situation in a healthy vascular wall, for instance, would it heal faster after mechanical injury?

A: Under normal conditions endothelium turns over slowly. In response to mechanical injury it was previously reported in classical studies from the seventies that re-endothelialization of denuded areas occur through proliferation and migration of arterial ECs from adjacent intact endothelium. The regeneration potential of local arterial ECs is, however, considered to be lower that the proliferation potential of stem cells in general. Since ECs over an atherosclerotic plaque are dysfunctional it would be reasonable to claim that a healthy vascular wall would heal faster after mechanical injury.

Q: Extending the last question, would it be important then to foster the health of the existing endothelium? Do you think that pharmacological interventions that improve endothelial function hasten recovery, e.g. after plaque disruption, which might be clinically relevant?

A: Although one should always be cautious about extrapolating from animal models to human disease, our data suggest that if the circulating cell populations in humans have a role in the development or progression of atherosclerosis, then it is not by differentiating into plaque endothelial cells. Instead, it seems as if it would be more relevant to foster the health of the existing endothelium as you suggest and that this could be relevant clinically.

Q: Are you extending your studies, what are you next steps? What should the research community take from you study and in which direction should it go?

A: Maybe most importantly, our study illustrates the necessity of demanding clear experimental proof for new theories. The EPC theory has been widely adopted also in the setting of atherosclerosis, but experimental studies have been lacking.

At the moment we are finishing up a study on allograft vasculopathy in which we use a new double-transplantation technique developed in our group, to be able to distinguish between cells migrating into allograft from the flanking vasculature and those homing from blood.

Q: A final, provoking question, but would you say we should not look so much into the stem cell research?

A: Recent research has shown that “endothelial progenitor cells” may promote angiogenesis and endothelial proliferation by paracrine mechanisms and now some have gradually redefined the term“endothelial progenitor cells” to include circulating "angiogenic" cells without a putative endothelial fate. This is very confusing, since the literally correct definition of a progenitor cell as an immature precursor cell capable of differentiating into a mature cell type in vivo. On the other hand, these paracrine mechanisms could be important in promoting a healthy endothelium.

 

Conclusion:

Thank you so, so much for your time and this interview. This is a really elegant study with important implications for future research and clinically. In case someone has further discussion points, may we forward them via my E-mail (herrmann.joerg@mayo.edu) or may they contact you directly at your E-mail address?

A: I indeed appreciate that you liked our paper and was honored to participate in this brief interview. If someone should have further points to ask or discuss they are very welcome to contact me directly at mette.hagensen@ki.au.dk.

References


Journal: Circulation. 2010 Feb 23;121(7):898-905. Epub 2010 Feb 8.

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.

Contact us

Working Group on Atherosclerosis & Vascular Biology

European Society of Cardiology

Les Templiers
2035 Route des Colles
CS 80179 BIOT

06903Sophia Antipolis, FR