Age-Associated Calcification: Insights from Murine Models

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Overall, this is a nicely done and very comprehensive review of aging models that explores the pathogenesis of aging and the potential connections with pathologic calcifications.  It tends to skew more toward explaining aging changes than necessarily linking these models with calcification mechanisms, but the table at the end partially reconciles this.  The role of age-related extracellular matrix changes,  particularly in diseases such as CPPD, cannot be ignored and little attention is paid to this in this review.  The discussion of atherosclerotic calcification in the first part of the paper does not mention the osteoblastic transdifferentiation that has recently been described by Linda Deemer's group.  

There are also a few minor issues.

1) The definition of the ANK protein on line 30 is not correct. 

2) Other mechanisms of ATP transport across the cell membrane are not addressed. 

Comments on the Quality of English Language

This needs minor editing for English.  For example, "hearth" is used in place of the word " heart" throughout this manuscript.  There are multiple other small mistakes

Author Response

Overall, this is a nicely done and very comprehensive review of aging models that explores the pathogenesis of aging and the potential connections with pathologic calcifications.  It tends to skew more toward explaining aging changes than necessarily linking these models with calcification mechanisms, but the table at the end partially reconciles this. 

1) The role of age-related extracellular matrix changes, particularly in diseases such as CPPD, cannot be ignored and little attention is paid to this in this review. 

We have added two sentences and two references "Finally, aging can affect cartilage matrix composition. ECM changes that occur during osteoarthritis, such as increased collagen I and reduced proteoglycans, may promote cartilage calcification ".

2)The discussion of atherosclerotic calcification in the first part of the paper does not mention the osteoblastic transdifferentiation that has recently been described by Linda Deemer's group. 

We have adressed this request in the paragraph 2.1, "Cardiovascular calcification, by adding the following sentence and reference "Finally, osteoblastic transdifferentiation of vascular smooth muscle cells seems to be a crucial mechanisms leading to vascular calcification and atherosclerosis during aging."

3) The definition of the ANK protein on line 30 is not correct. 

To our knowledge, the role of ANK has been recently revisited. Indeed, it appears to be an ATP channel rather a PPi channel. We have added the reference in the text "The Mineralization Regulator ANKH Mediates Cellular Efflux of ATP, Not Pyrophosphate".

4) Other mechanisms of ATP transport across the cell membrane are not addressed. 

We thank the reviewer for this point. We have added a paragraph and a reference at line 32 "ATP can also be released through various membrane transporters, such as pannexins, connexins, , and others, or via exocytosis. The principal supplier of ATP likely varies among tissues and remains unknown"

Reviewer 2 Report

Comments and Suggestions for Authors

The authors present a review on murine models of ectopic calcification, covering a variety of organ systems and animal models. I have several suggestions:

Section 2, Tissues affected by pathologic calcification. This section would benefit from a discussion of the specific types of calcium crystals that lead to CAVD, myocardium, arteries, calcific tendinopathy, etc. Please include and cite cadaveric/histopathologic studies.

Section 2.2, Musculoskeletal calcification. The final paragraph of this section focuses on articular cartilage. As above, this section would benefit from distinguishing between BCP and CPP crystals, e.g., the statement “present in 100% of cartilage samples at the time of joint replacement” refers to BCP but not to CPP. There are a number of cohort studies focused on the relationship between CPP and OA progression (Neogi, et al; Latourte, et al.; others) and these provide conflicting results. Rosenthal and Ryan (NEJM 2016) discuss the possible consequences of CPP crystals for cartilage and this should be included and elaborated upon.

Page l, line 26:  I believe the correct term is “calcium pyrophosphate dihydrate (CPP)”, not “dehydrate”

Please edit for English grammar throughout. For example, page 1 line 20 should read “128 million people”, not “128 millions of people”; line 24 “heart valves” not “hearth valves”

Figure 1 shows BCP crystals forming in a matrix vesicle. Rosenthal & Ryan NEJM 2016 figure and text describe CPP crystals forming in articular cartilage vesicles. Please update Figure 1 accordingly.

Comments on the Quality of English Language

Requires some editing.

Author Response

The authors present a review on murine models of ectopic calcification, covering a variety of organ systems and animal models. I have several suggestions:

1) Section 2, Tissues affected by pathologic calcification. This section would benefit from a discussion of the specific types of calcium crystals that lead to CAVD, myocardium, arteries, calcific tendinopathy, etc. Please include and cite cadaveric/histopathologic studies.

We have now added a sentence on the kind of crystals found in the different calcification diseases, and the corresponding references "While a mixture of BCP and CPP crystals have been found in cartilage, in other pathologically calcifying tissues BCP crystals are prominent."

2) Section 2.2, Musculoskeletal calcification. The final paragraph of this section focuses on articular cartilage. As above, this section would benefit from distinguishing between BCP and CPP crystals, e.g., the statement “present in 100% of cartilage samples at the time of joint replacement” refers to BCP but not to CPP. There are a number of cohort studies focused on the relationship between CPP and OA progression (Neogi, et al; Latourte, et al.; others) and these provide conflicting results. Rosenthal and Ryan (NEJM 2016) discuss the possible consequences of CPP crystals for cartilage and this should be included and elaborated upon.

We thanks the reviewer for the suggestions. We have made the following changes in the text, and added references accordingly:

"Two different types of calcium-containing crystals have been found in cartilage: BCP and CPP crystals. BCP crystals, rather than CPP, appear prevalent in OA, and are present in 100% of cartilage samples at time of joint replacement".

"The consensus on the role of crystals in OA remains controversial. While some studies suggest they are not etiologically significant, recent clinical and experimental data support their contribution to OA development and progression. Radiographically detected mineralization has been linked to an increased risk of knee pain over two years in individuals both under 60 years old and over 60 years old . Additionally, cartilage calcification has been associated with increased cartilage and meniscus degeneration over a four-year period . "

3) Page l, line 26:  I believe the correct term is “calcium pyrophosphate dihydrate (CPP)”, not “dehydrate”

This has been fixed

4) Please edit for English grammar throughout. For example, page 1 line 20 should read “128 million people”, not “128 millions of people”; line 24 “heart valves” not “hearth valves”

This has been fixed

5) Figure 1 shows BCP crystals forming in a matrix vesicle. Rosenthal & Ryan NEJM 2016 figure and text describe CPP crystals forming in articular cartilage vesicles. Please update Figure 1 accordingly.

We have updated Figure 1 according to the reviewer's suggestion.

Round 2

Reviewer 2 Report

Comments and Suggestions for Authors

The authors have addressed my comments.

Comments on the Quality of English Language

This is improved.