Letter by Kumar Regarding Article, “Contribution of Intimal Smooth Muscle Cells to Cholesterol Accumulation and Macrophage-Like Cells in Human Atherosclerosis”
To the Editor:
I recently read “Contribution of Intimal Smooth Muscle Cells to Cholesterol Accumulation and Macrophage-Like Cells in Human Atherosclerosis” by Allahverdian et al,1 published in Circulation. In my opinion, the article, in addition to the limitations highlighted in the accompanying editorial,2 has some methodological flaws that merit discussion.
First, the authors do not show any difference in cholesterol accumulation between early and advanced plaques, which, in my opinion, would be essential to justify their study.
The authors state that mouse and human atherosclerotic vessels are not similar, which is well accepted by several others involved in atherosclerosis research.3 Despite this, the authors of this study1 use a lipid fixation method developed for mouse tissue to process the human tissue. How valid is this method for processing human tissue samples? In my opinion, it would be appropriate to validate the relevance of the mouse tissue protocol’s application in processing human tissue. Without such validated protocols the data can be misleading.
In relation to Figure 1 of this article,1 in my opinion, it is not accurate to conclude on extra- and intracellular lipid in arterial sections based on 2-dimensional immunostaining images. Often, signals from the cell membrane surface can be falsely concluded as intracellular in the 2-dimensional imaging.
Further, in my opinion, making a conclusion on the origin of foam cells just based on the immunostaining imaging method without proper cell labeling/genetic tracking protocols is not only inappropriate (especially when the techniques to perform such rigorous studies are available), but it can also lead to the danger of data overinterpretation and false-positive conclusions.
Immunostaining is a semiquantitative approach and is extensively subjective, especially in the absence of sections stained with isotype control. It is unfortunate that many researchers do not present isotype control because of space constraints, which, in my opinion, is not a good practice because isotype controls are valuable in interpreting data and ruling out false-positive findings. Moreover, a relatively nonsubjective and quantitative approach such as fluorescence-activated cell sorting should preferably be adopted as a second method to substantiate the immunostaining findings.
The authors inappropriately use the term intima; I presume that neointima would be more appropriate term.
On pages 1554 to 1555, the authors indicate that “ABCA1 expression is reduced in SMCs in the intimal layer in comparison with the medial layer of human coronary arteries, providing a potential reason for increased SMC foam cell formation in the intima.” In my opinion, quantitative data on this parameter would be more objective in supporting this statement. Moreover, biochemically, the expression levels of a protein are not indicative of its activity; hence, without looking at the ATP-binding cassette transporter A1 (ABCA1) activity, it may be misleading to make a conclusion on the pathophysiological role of the protein. There are several examples in the literature to support that protein expression levels often do not correlate with its activity levels. Nevertheless, the differential expression of ABCA1 in smooth muscle versus monocytes/macrophages at various stage of plaque is an interesting finding, which can be further explored.
Although this study does indicate that cells staining for a smooth muscle marker have higher expression of ABCA1 in early versus advanced plaques, whereas cells staining for CD45 have a higher expression of ABCA1 in advanced versus early plaques, concluding anything beyond this, in my opinion, would be overinterpretation, not supported with rigorous scientific evidence and definitely insufficient to make a conclusion on the origin of cells in the neointima.
Arun H.S. Kumar, DVM, PhD
School of Veterinary Medicine
University College Dublin
Belfield, Dublin, Ireland
- © 2015 American Heart Association, Inc.
- Allahverdian S,
- Chehroudi AC,
- McManus BM,
- Abraham T,
- Francis GA
- Fisher EA,
- Miano JM
- Stylianou IM,
- Bauer RC,
- Reilly MP,
- Rader DJ