Comments for:

---

Response and clarifications

Submitter: Ariela Benigni | ariela.benigni@marionegri.it

Mario Negri Institute for Pharmacological Research

Published November 11, 2009

In response to the letter from Dr. Amorena, we offer the following as clarification.

1. Survival in the two groups of animals is given in figure 1 from which survival of each animal can be inferred. The sentence in the text could have been “These remaining mice lived longer up to additional 7 months”.
2. Overall this was a survival study and autopsies have not been performed. We therefore have no data on the cause of death. We know from other studies that mice of the same genetic background as ours mostly die of intestinal lymphoma (Toxicol Pathol, 2001, 29(6): 653). Criteria to establish imminent death for those animals which have been sacrified were taken from the “Guidance document on the recognition, assessment, and use of clinical signs as humane endpoints for experimental animals used in safety evaluation” November 2000. Organisation for Economic Co-operation and Development (OECD), Environmental Health and Safety Publications, Series on Testing and Assessment, No. 19. http://www.olis.oecd.org/olis/2000doc.nsf/LinkTo/NT00002E46/$FILE/00087372.PDF.
3. Constant heart weight - evaluated on fresh tissue - in the presence of the reported changes (Fig3) can possibly be explained by a dysfunctional endothelium of KO animals leading to a disturbed water permeability, which might be even compatible with a preserved cardiac function as recently documented (Butler TL et al, Thorac Cardiovasc Surg, 138(1): 141-7, 2009). Here we see two possible mechanisms: i. compensatory activation in KO mice of an angiotensin II receptor subtype, which may interfere with vascular hydraulic permeability ii. related changes in the VEGF/eNOS signaling also known to disturb endothelial permeability. In the absence of data, we preferred not to speculate at this stage. Studies are undergoing in the lab to test these hypotheses and results will be the subject of a subsequent publication.
4. Panels A and C of figure 4 have been taken at the same magnification (20x) which definitely allows to compare photographs in WT and AT1A -/- mice. Panel B is a detail of panel A taken at a slightly higher magnification to better appreciate intraparietal accumulation of foam cells in WT animals.

We are grateful to Dr. Amorena and his colleagues for their interest in our study.

---

Concerns about the AT1a^-/- longevity paper

Submitter: Carlos Amorena | camorena@unsam.edu.ar

Authors: Moretta R., García-Gras E., Tiscornia G., Argenziano M., Potilinski C., Siciliano E.

CESyMA-UNSAM

Published November 2, 2009

We have read the paper “Disruption of the Ang II type 1 receptor promotes longevity in mice” (JCI,2009,119:524-530) by Benigni et al. We think that there are points in this paper that deserve consideration.

The first result is presented ambiguously. The text reads: “At 29 months, when all wild-type animals died, 17 AT1a-deficient mice (85%) were still alive. These remaining mice lived for an additional 7 months.” However from Figure 1 we see that the remaining 17 mice were not alive at month 36. Furthermore, there is no indication as to whether the mice used to determine life span died spontaneously or were sacrificed when death appeared imminent. The second option is actually mentioned for other experiments in the methods section, but there is no reference to the criteria used to determine imminent death. In the list of signs that point to the proximity of death used by Jackson laboratory (http://research.jax.org/faculty/harrison/ger1vLifespan1.html) each one of them emphasize the external bad condition of mice. Assuming that this list of signs was used, it is remarkable that the internal organs of the AT1a^-/- mice were in such good conditions as it is depicted. One could wonder what were the animals dying of.

Another subject that deserves comment is related to the differences the authors observed between WT and AT1a^-/- mice in regard to cardiac hypertrophy. Even though it is not reported, we can reason that if body weight didn’t change (Figure 2A) and neither did heart weight/body weight ratio (Figure 3A), heart weight should remain constant between the two strains. Plus, the number of myocytes is also constant (Figure 3B). Then, how come is it possible that crossectional area of cardiomyocytes (Figures 3C) and interstitial collagen (Figure 3D) are statistically different? We wonder where the surplus mass originated in the larger fibers is placed. But this remarkable find isn’t even discussed.

Figure 4 shows the histology of the aorta of WT and AT1a-/- mice. Relying on the size of the nuclei in wild type vs. AT1a^-/-, it looks like the magnification of the aorta of the wild type differs from that of the AT1a^-/- animals. We find this confusing, for it prevents the observer from comparing the photographs properly. The legend “Original magnification, 20X”, without a scale bar seems to imply that the magnification is the same in all cases. We thank the Journal for the opportunity to discuss this issue.