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. 2020 Jul 2;15(7):e0235553. doi: 10.1371/journal.pone.0235553

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation

Batmunkh Bumdelger 1,#, Mikage Otani 1,#, Kohei Karasaki 1, Chiemi Sakai 1, Mari Ishida 1, Hiroki Kokubo 1,*, Masao Yoshizumi 1,*
Editor: Helena Kuivaniemi2
PMCID: PMC7331998  PMID: 32614927

Abstract

Aortic aneurysm refers to dilatation of the aorta due to loss of elasticity and degenerative weakening of its wall. A preventive role for osteoprotegerin (Opg) in the development of abdominal aortic aneurysm has been reported in the CaCl2-induced aneurysm model, whereas Opg was found to promote suprarenal aortic aneurysm in the AngII-induced ApoE knockout mouse aneurysm model. To determine whether there is a common underlying mechanism to explain the impact of Opg deficiency on the vascular structure of the two aneurysm models, we analyzed suprarenal aortic tissue of 6-month-old ApoE-/-Opg-/- mice after AngII infusion for 28 days. Less aortic dissection and aortic lumen dilatation, more adventitial thickening, and higher expression of collagen I and Trail were observed in ApoE-/-Opg-/- mice relative to ApoE-/-Opg+/+ mice. An accumulation of α-smooth muscle actin and vimentin double-positive myofibroblasts was noted in the thickened adventitia of ApoE-/-Opg-/- mice. Our results suggest that fibrotic remodeling of the aorta induced by myofibroblast accumulation might be an important pathological event which tends to limit AngII-induced aortic dilatation in ApoE -/-Opg-/- mice.

Introduction

Aortic aneurysm (AA) refers to a dilatation of the aorta due to loss of elasticity and degenerative weakening of its wall. Continuous expansion of the aorta results in rupture and is associated with a high mortality rate [1]. Analyses of AA in experimental animal models, including the CaCl2-induced mouse model and the AngII-induced ApoE knockout (KO) mouse model [2, 3], are important for understanding the pathogenesis of this disease and for developing effective drug treatments aimed at arresting aortic expansion [4–7].

Osteoprotegerin (Opg, also referred to as TNFRSF11B), a member of the tumor necrosis factor (TNF) receptor superfamily, functions as a decoy receptor to regulate various factors across many biological processes [8]. For example, Opg has been shown to regulate bone metabolism through the Receptor activator of nuclear factor kappa-B ligand (Rankl) [9, 10] and apoptosis of cancer cells through TNF-related apoptosis-inducing ligand (Trail). Given that vascular diseases are often involved in bone pathologies [11, 12], and because Opg is expressed in vascular smooth muscle cells (VSMCs) [13] and serum levels of OPG are elevated in cardiovascular disease [14–17], there is great interest in the roles Opg may play in the vascular system.

We recently reported that Opg plays a preventive role in the development of abdominal AA (AAA) in the CaCl2-induced aneurysm model [18]. In Opg KO mice, we found larger aneurysms with destruction of the aortic medial layer, which had increased expression of matrix metalloproteinase (Mmp)-9 and Trail. The expression of Opg in aortic tissue was also increased in response to aneurysm induction in wild-type mice. We concluded in this recent study that Opg prevents AAA formation through its antagonistic effect on Trail. However, another group reported that Opg can promote the development of aneurysms in the suprarenal aorta (SRA) in the AngII-induced ApoE KO mouse model [19]. The authors of that study attributed the lower incidence of aneurysms and rupture in ApoE-/-Opg-/- mice to the down-regulation of proteolytic enzyme expression. The two reports discussed above suggest opposing results for aneurysm formation in Opg-deficient mice. Interestingly, a recent study found that low dose Opg had a preventive effect in both AAA models, although the mechanism underlying the preventive effect is unclear [20].

In the present study, we found a lower incidence of aortic dissection and a lower tendency for aortic dilatation in Opg-deficient mice. There were no structural differences in medial elastic fibers between ApoE-/-Opg-/- and ApoE-/-Opg+/+ mice. However, more adventitial thickening and increased expression of collagen I, α-smooth muscle actin (SMA), vimentin, and Trail were observed in ApoE-/-Opg-/- mice relative to ApoE-/-Opg+/+ mice. This suggests that fibrotic remodeling of the aorta may have resulted from an accumulation of myofibroblasts in ApoE-/-Opg-/- mice, which was also previously observed in the CaCl2-induced AAA model [18]. Our results suggest that Opg deficiency may lead to fibrotic remodeling of the aorta, possibly enhanced by Trail signaling, and is an important pathological event that tends to limit AngII-induced SRA dilatation and dissection in ApoE KO mice.

Results

Opg deficiency tends to limit AngII-induced aortic dissection and dilatation

In order to confirm the phenotypic differences in the two aneurysm models resulting from Opg deficiency, we crossed Opg KO mice with ApoE KO mice to generate ApoE-/-Opg-/- mice. The Opg KO mice were then treated with AngII to promote aneurysm development in the suprarenal aorta (SRA) (S1A Fig in S1 File). Although Moran et al. reported a significantly smaller median maximum diameter of the SRA in ApoE-/-Opg-/- mice compared to that in ApoE-/-Opg+/+ mice, we did not observe a significant reduction in the maximum external diameter in ApoE-/-Opg-/- mice relative to ApoE-/-Opg+/+ mice (Fig 1A and 1B, S2A Fig–S2C Fig in S1 File). Moreover, no significant differences were observed in survival rate and concentrations of serum cholesterol between ApoE-/-Opg-/- and ApoE-/-Opg+/+ mice at 28 days after AngII infusion (S2F Fig and S2G Fig in S1 File). However, on visual inspection, the diameter of the SRA in ApoE-/-Opg-/- mice appeared to be somewhat smaller, and multiple hematomas were observed in the SRA of ApoE-/-Opg+/+ mice. Thus, we adopted Daugherty’s classification system for aortic aneurysms [21] and categorized the mice into three groups based on dilatation of the SRA and the presence of visible hematoma (No Aneurysm, Aneurysm, and Dissection groups). In ApoE-/-Opg+/+ mice, 75% of SRAs were dilated and the majority of them had dissected (Fig 1C). However, in ApoE-/-Opg-/- mice, 50% of SRAs were dilated and only a few had dissected. This suggests that Opg deficiency may have a preventive effect on AngII-induced aortic dilatation and dissection (43.75% in ApoE-/-Opg+/+ mice vs. 18.75% in ApoE-/-Opg-/- mice).

Fig 1. Opg deficiency tends to suppress AngII-induced aortic aneurysms.

Fig 1

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(A) Aortas of AngII-infused ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice were categorized into three groups based on diameter and the presence of visible hematoma (No Aneurysm, Aneurysm, and Dissection groups). Scale bars indicate 1 mm. (B) External diameter of the SRA in AngII-infused ApoE-/-Opg+/+ (gray, n = 16) and ApoE-/-Opg-/- (white, n = 16) mice. All measurements are shown as box plots and each measurement is shown as a white circle (No Aneurysm), gray triangle (Aneurysm), or black cross (Dissection). (C) The incidence (%) of aortic aneurysms is shown for No Aneurysm (white), Aneurysm (gray), and Dissection (black) groups. Statistical significance: p<0.05.

Opg deficiency results in aneurysm with adventitial thickening

Since Opg deficiency may have a preventive effect on aortic dilatation and dissection, transverse cross-sections of the SRA were used to measure the size of the aortic lumen and evaluate the aortic wall structure, including the medial and adventitial layers. HE staining revealed narrower aortic lumens and smaller dissecting hematomas in ApoE-/-Opg-/- mice compared to ApoE-/-Opg+/+ mice (Fig 2A(HE)). The area of the aortic internal lumen tended to be smaller in ApoE-/-Opg-/- mice compared with ApoE-/-Opg+/+ mice, although the difference was not significant (Fig 2B).

Fig 2. Opg deficiency tends to suppress AngII-induced aortic dilatation and promotes adventitial thickening.

Fig 2

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(A) Representative cross sections of the aorta stained with HE, EVG, and AZAN. Areas selected by boxes surrounded by a black dotted line are magnified below. Scale bars indicate 0.1 mm. (B) Internal area of the suprarenal aorta (SRA) of ApoE-/-Opg+/+ (n = 16) and ApoE-/-Opg-/- (n = 16) mice. Measurements for all samples are presented as box plots. Measurements for the three groups are presented as bar graphs. n = 4 and 8 for the No-aneurysm group, n = 5 and 5 for the Aneurysm group, and n = 7 and 3 for the Dissection group in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. (C) Medial layer width of the SRA of AngII-infused mice in each group. N.S; not significant. (D) Relative adventitial area of the SRA of AngII-infused mice in each group. Statistical significance: p<0.05.

There were no differences in the structure of medial elastic fibers in the Aneurysm group between ApoE-/-Opg-/- and ApoE-/-Opg+/+ mice, as assessed by EVG and AZAN staining (Fig 2A (panels EVG and Aneurysm), S3A Fig in S1 File). In the Dissection group, a complete disappearance of medial elastic fibers in which atherosclerotic plaques invaded the disrupted side of the aortic circumference was observed in mice of both genotypes (Fig 2A (panels EVG and Aneurysm)). There was no significant difference in the width of the medial layer between mice of both genotypes (Fig 2C). However, significant adventitial thickening and accumulation of collagen were noted in the Aneurysm and Dissection groups in ApoE-/-Opg-/- mice (Fig 2A (panel AZAN), Fig 2D, S3B Fig in S1 File). These observations suggest that adventitial thickening might be associated with the smaller aortic diameter and lower tendency of dissection in ApoE-/-Opg-/- mice.

Fibrotic remodeling of the SRA and accumulation of myofibroblasts in ApoE-/-Opg-/- mice

Given the accumulation of collagen in ApoE-/-Opg-/- mice, we examined the type of collagen that accumulated in the adventitia. Collagen I, but not collagen III, accumulated in ApoE-/-Opg-/- mice following AngII infusion (Fig 3A, S4A Fig and S4B Fig in S1 File). The area of collagen I expression was significantly larger in ApoE-/-Opg-/- mice compared to that in ApoE-/-Opg+/+ mice (Fig 3B). As expected, no differences were found in collagen I expression between ApoE-/-Opg-/- and ApoE-/-Opg+/+ mice in the H2O-infused controls (S4C Fig in S1 File). Since TGF-β1 is the central mediator of fibrogenesis [22], we examined its mRNA expression in SRA tissue and found it to be significantly higher in ApoE-/-Opg-/- mice compared to ApoE-/-Opg+/+ mice after AngII infusion (Fig 3C). This result further supports the finding that Opg deficiency promotes fibrotic remodeling of the aorta.

Fig 3. Collagen accumulation in aortas of ApoE-/-Opg-/- mice.

Fig 3

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(A) Representative immunofluorescence images of aortas of AngII-infused mice stained with an anti-collagen I antibody. Boxed areas (with white dotted lines) are magnified below. Scale bars indicate 100 μm. (B) Percent area of collagen I expression in aortas of ApoE-/-Opg+/+ (n = 16) and ApoE-/-Opg-/- (n = 16) mice. Measurements for all samples are presented as box plots. Measurements for the three groups are presented as bar graphs (n = 4 and 8 for the No Aneurysm group, n = 5 and 5 for the Aneurysm group, and n = 7 and 3 for the Dissection group in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively). (C) Tgf-β1 mRNA expression in the SRA at days 0, 7, and 28 after initiation of AngII infusion in ApoE-/-Opg+/+ (n = 4, 5, 5) and ApoE-/-Opg-/- (n = 5, 5, 5) mice. Statistical significance: p<0.05.

To determine the types of cells which accumulated in the adventitia, immunohistochemistry was performed using anti-SMA and anti-vimentin antibodies to mark smooth muscle cells and fibroblasts, respectively. In ApoE-/-Opg-/- mice, both SMA and vimentin were distributed in the adventitia, especially in the Aneurysm and Dissection groups (Fig 4A and 4B). Since both SMA and vimentin are known to be expressed in myofibroblasts, the accumulated cells that over-express collagen I in the adventitia of ApoE-/-Opg-/- mice are likely myofibroblasts. Interestingly, these myofibroblasts were found not only in the adventitia, but also in the outer layers of the media in ApoE-/-Opg-/- mice. Medial cells expressing SMA but not vimentin in ApoE-/-Opg+/+ mice were unlikely to be myofibroblasts (Fig 4A). Next, we measured the expression of matrix metalloproteinases (MMPs) in SRA tissue, but found no difference between ApoE-/-Opg-/- and ApoE-/-Opg+/+ mice after AngII infusion (S5A Fig-S5C Fig in S1 File). These findings collectively suggest that accumulation of myofibroblasts could be one of the causes of adventitial thickening in ApoE-/-Opg-/- mice after AngII infusion.

Fig 4. Accumulation of myofibroblasts in adventitias of AngII-infused ApoE-/-Opg-/- mice.

Fig 4

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(A) Representative double-immunofluorescence images of aortas after AngII infusion with anti- SMA (green) and anti-vimentin (red) antibodies. Scale bars indicate 100 μm. Areas selected by the white box (dotted line) are magnified below. Nuclei were stained with DAPI (blue). Scale bars indicate 25 μm. Medial layer (m), Adventitial layer (a), hematoma (b). The two arrowheads show borders of the medial layer. The white dotted line in the magnified panels indicates the border of the medial and adventitial layers. (B) Percent area of SMA expression in adventitias of ApoE-/-Opg+/+ (n = 16) and ApoE-/-Opg-/- (n = 16) mice. Measurements for all samples are presented as box plots. Measurements for the three groups are presented as bar graphs. n = 4 and 8 for the No Aneurysm group, n = 5 and 5 for the Aneurysm group, and n = 7 and 3 for the Dissection group in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. Statistical significance: p<0.05.

Up-regulation of Trail in SRAs of AngII-infused ApoE-/-Opg-/- mice

To understand how Opg deficiency contributes to adventitial thickening, we examined the expression of Trail in aneurysm tissue of AngII-infused ApoE-KO mice, given the known role of Opg as a decoy receptor for Trail [8]. Trail expression was up-regulated in the adventitia and outer layers of the media in ApoE-/-Opg-/- mice after AngII infusion (Fig 5A and 5C), but not in ApoE-/-Opg+/+ mice or after H2O infusion (S5D and S5F Fig in S1 File). Notably, Trail was mainly expressed in cells that were double positive for SMA and vimentin. These cells were also positive for Ki67, a cell proliferation marker (Fig 5B), implying that Trail induces the proliferation of these cells autonomously [23].

Fig 5. Trail upregulation in aneurysm tissue of AngII-infused ApoE-/-Opg-/- mice.

Fig 5

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(A) Representative immunofluorescence images of aortic tissue sections from AngII-infused mice stained with anti-Trail (green) antibody. Scale bars indicate 100 μm. Areas selected by the white box (dotted line) in the Aneurysm group are magnified below. (B) Magnified immunofluorescence images of aortic tissue sections from the AngII-infused Aneurysm group stained with anti-Trail (green; a, f), anti-SMA (green; b, g), anti-vimentin (red; c, h), anti-Ki67 (red; d, i), and anti-F4/80 (red; e, j) antibodies in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice. Nuclei were stained with DAPI (blue; c-DAPI, h-DAPI). Scale bars indicate 25 μm. Medial layer (m), adventitial layer (a). The two arrowheads show borders of the medial layer. The white dotted line in the magnified panels indicates the border of the medial and adventitial layers. Small arrows in panel j indicate large round-shaped macrophages infiltrating the adventitia. Statistical significance: p<0.05. (C) Percent area of Trail expression in medial and adventitial layers of ApoE-/-Opg+/+ (n = 16) and ApoE-/-Opg-/- (n = 16) mice. Measurements for all samples are presented as box plots. Measurements for the three groups are presented as bar graphs. n = 4 and 8 for the No Aneurysm group, n = 5 and 5 for the Aneurysm group, and n = 7 and 3 for the Dissection group in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice, respectively. Statistical significance: p<0.05.

Trail has also been reported to promote chemotactic migration of not only SMCs, but also monocytes towards the site of inflammation [24]. Round shaped, F4/80 macrophages were detected in the adventitia of ApoE-/-Opg-/- mice (Fig 5B; S5G Fig in S1 File), suggesting that increased Trail expression may lead to more inflammation, myofibroblast accumulation, and fibrotic remodeling of the aortic wall in Opg-deficient mice.

Discussion

Consistent with results reported by Moran et al. [19], we found that Opg deficiency tended to limit AngII-induced aortic dissection and dilatation. Gross phenotypic differences are evident between the CaCl2-induced aneurysm model and AngII-induced aneurysm model [18, 19]. However, we observed adventitial thickening accompanied by myofibroblast accumulation and increased expression of collagen I and Trail in aneurysm tissue of ApoE-/-Opg-/- mice. These pathological and cellular changes of the aortic wall were also reported in the CaCl2-induced aneurysm model [18], suggesting that adventitial thickening with myofibroblast accumulation could be a common feature of Opg deficiency.

Myofibroblasts in aortic tissue promote the excessive production of extracellular matrix, including collagen, and subsequent fibrotic remodeling of the aorta [25]. Disorders of collagen fiber assembly are thought to underlie aortic dilatation and dissection. For example, weakness in connective tissue caused by mutations in various types of collagen and enzymes involved in collagen maturation in Ehlers-Danlos syndrome results in aortic aneurysms [26]. Moreover, Dobrin et al. reported that degradation of collagen, rather than elastin, is an important cause of aneurysm development, suggesting that collagen fibers protect the aorta from internal pressure-induced expansion [27]. When applied to the present study, these findings suggest that excessive collagen accumulation in the adventitia may have strengthened the aortic tissue. This in turn could have resulted in smaller aneurysms and a lower rate of dissection events in ApoE-/-Opg-/- mice in the AngII-induced aneurysm model. In light of studies reporting that Trail induces collagen I transcription in fibroblast cells [28], Trail could potentially play a role in collagen accumulation in myofibroblasts. Fibrotic remodeling of the aorta by myofibroblasts might protect against AngII-induced aneurysm and dissection in ApoE-/-Opg-/- mice.

In our previous study, which used the CaCl2-induced aneurysm model, Opg deficiency exacerbated AAAs, despite adventitial thickening, increased Trail expression, and the appearance of myofibroblasts [18]. We speculate that the contrasting phenotypes of the AngII-induced and CaCl2-induced aneurysm models could be due to different processes involved in aneurysm formation. In the AngII-induced ApoE KO mouse aneurysm model, local medial destruction is observed only in the plaque area. It is currently thought that aneurysms and dissection start at small cleavages which develop after infusion of AngII, leading to local destruction of the medial layer [29]. Concurrently, collagen accumulation in the adventitia induced by AngII infusion in ApoE-/- /Opg-/- mice limits the development of aneurysms and dissection. On the other hand, in the CaCl2-induced model, complete destruction of the medial layer is observed in a broad area. This extensive destruction of the medial layer may promote enlargement of the aortic lumen. Increased Trail expression, due to the absence of Opg, up-regulates the expression of proteolytic enzymes, including Mmp9 and Mmp2, resulting in an acceleration of elastic medial tissue destruction. Although adventitial thickening simultaneously progresses after CaCl2 treatment in Opg KO mice, it may be insufficient to compensate for the severe and extensive medial tissue destruction and limit aortic enlargement.

In conclusion, adventitial thickening with collagen accumulation induced by AngII infusion may increase the strength of aortic tissue and potentially limit the development of aortic aneurysms and dissection in ApoE-/-Opg-/- mice. At the same time, aortic tissue may lose flexibility in elastic vessels, thereby increasing cardiac load. AngII infusion in the absence of Opg may promote excessive reactions in aortic tissue, suggesting that Opg plays a potential role in aortic tissue homeostasis and maintenance of proper blood pressure. In the therapeutic context, targeting aortic tissue with agents that induce adventitial thickening with collagen accumulation may help suppress dissection events. Such treatments may include focal drug delivery systems in which an anti-Opg antibody is delivered to plaques, or direct application of drugs to stent grafts which would allow for focal induction of adventitial thickening.

Materials and methods

Mice

Opg KO (ApoE+/+/Opg-/-) mice of the C57BL/6J strain background (CLEA Japan, Inc.) were crossed with ApoE KO (ApoE-/-/Opg+/+) mice to obtain ApoE+/-Opg+/- mice. Progeny were inter-crossed to establish an ApoE-/-Opg+/- line, which was then inter-crossed to generate ApoE-/-Opg-/- mice and control ApoE-/-Opg+/+mice. Genotypes were determined by PCR (94°C for 30 sec; 55°C for 30 sec and 72°C for 30 sec for 35 cycles) using the following primers: forward primer 5’-CTG ACC ACT CTT ATA CGG AC AG-3’and reverse primer 5’-CTA AGT TAG CTG CTG TCT GGC-3’ for the Opg-wild type allele; forward primer 5’-CTG ACC GCT TCC TCG TGC TTTAC-3’ and the above-mentioned reverse primer for the Opg-mutant allele; forward primer 5’-ACT CTA CAC AGG ATG CCT AGC-3’ and reverse primer 5’-CTC ACG TCA GTT CTT GTG TGAC-3’ for the ApoE-wild type allele; and the above-mentioned forward primer and reverse primer 5’-GCC GCC CGA CTG CAT CT-3’ for the ApoE-mutant allele.

Aortic aneurysm model

Six-month-old male ApoE-/-Opg+/+ (n = 16) and ApoE-/-Opg-/- (n = 16) mice were infused with AngII as described previously [30]. Under anesthesia, a micro-osmotic pump (ALZET Model 1004, Durect Corporation) was implanted into the subcutaneous space left of the dorsal midline for infusion of AngII (Sigma-Aldrich) or H2O at a rate of 1.0 μg/kg/min for 28 days. Mice were sacrificed by anesthetia overdose. After cutting the sternum and exposing the thoracic cavity, blood from the right ventricle was collected for storage during perfusion with PBS. Mice were then perfused with 4% paraformaldehyde. Whole aortas were excised for morphometric analysis and measurement of SRA diameters. Images were taken of whole aortas to measure the external diameter of SRAs. SRAs were then separated from aortas and embedded in paraffin for subsequent histological analysis.

For RNA isolation, aortas were harvested after perfusion with PBS and stored in RNA-later solution (Ambion). This experiment was approved by the Committee of Animal Experimentation at Hiroshima University (A08-32) and carried out in accordance with the approved protocol. All surgeries were performed under a combination of three anesthetic solutions (medetomidine, butorphanol, and midazolam), and efforts were made to minimize suffering during and after surgery.

Morphological examination and measurement of aortic diameters

The maximum external diameter of the SRA as shown in S1A Fig in S1 File was measured (Fig 1B). The external diameter of the SRA in the H2O-infused group (S2C Fig in S1 File) was used as a control to categorize AngII-infused aortas into three groups based on maximum external diameter and the presence of visible hematoma (No-aneurysm group: diameter less than 1.5 times the H2O-infused control; Aneurysm group: diameter more than 1.5 times the H2O-infused control without visible hematoma; Dissection group: diameter more than 1.5 times the H2O-infused control with visible hematoma). Paraffin-embedded aortic tissues were used to generate 6 μm-thick sections, which were cut at the level of the SRA. The tissue sections were then subjected to Hematoxylin/Eosin (HE), Elastica van Gieson (EVG), and Analine Blue (AZAN) staining. The sections were used to measure the internal area of the SRA, the width of the medial layer, and the relative area of the adventitia. Borders of the aortic lumen were marked with a dotted circle (S1B Fig in S1 File) and used to measure the internal area of the SRA (Fig 2B). Red lines show 5–8 measurements of the medial layer width on the side of the aortic circumference which was not disrupted by atherosclerotic plaques (S1B Fig in S1 File). Average values were used (Fig 2C). The total area (TA) of the aorta included all aortic layers. The arterial area (AA), which included the lumen, endothelium, and media, and the bleeding area (BA), which included hematoma between medial and adventitial layers, were marked by dotted circles (S1C Fig and S1D Fig in S1 File). After measuring each area, the relative area of the adventitia was calculated using the following equation: TA-AA-BA/TA. The % area of collagen I expression was calculated by dividing the signal area by the entire aortic area (excluding the hematoma area) (Fig 3B). The % area of SMA expression was calculated by dividing the signal area by the area of the adventitial layer (Fig 4B). The % area of Trail expression was calculated by dividing the signal area by the area of medial and adventitial layers (Fig 5C). Area selection was performed using Photoshop software and was confirmed by three independent researchers. Area measurements were performed using Image-J software (NIH, USA).

Immunohistochemistry (IHC)

Sections were pre-incubated in antigen retrieval solution (pH 5.2) at 90°C for 45 minutes, based on the manufacturer’s instructions (Dako), and then blocked with 1% bovine serum albumin in 0.1% Tween-phosphate buffered solution (PBS) for 1 hr. After pre-treatment and blocking, sections were incubated with a primary antibody overnight at 4°C and subsequently with appropriate secondary antibodies for 2 hr at room temperature, followed by counterstaining with 4’-6-diamidino-2-phenylindole (DAPI). After primary antibodies, listed in Table 1, sections were treated with anti-mouse, -rat, or -rabbit antibodies conjugated wiith Alexa Fluor 488 or 555 dye (donkey, 1:500; Life Technologies). Signals were detected using a DMI4000 fluorescence microscope (Leica Microsystems).

Table 1. List of antibodies.

Specificity Vendor Cat# Lot# dilution
Collagen I Abcam ab21286 GR46228-1 1:250
Collagen III Abcam ab7778 GR52659-1 1:250
TRAIL Abcam ab2435 GR14018-5 1:50
Actin, α-Smooth Muscle (Monoclonal clone 1A4) Sigma-Aldrich A2547 032M4822 1:200
Vimentin BioVision 3634 1:50
Ki67 Abcam ab15580 GR101835-1 1:100
F4/80 (BM8) Santa Cruz sc-52664 B1810 1:50
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Quantitative Real-time PCR

Total RNA was isolated using TRIzol reagent (Invitrogen). Reverse transcription was performed using the ReverTra Ace qPCR RT Kit (TOYOBO). Real-time PCR was conducted using SYBR Premix Ex Taq II (Takara Bio Inc. and Kapa Biosystems Inc.). Intensities of PCR products using primers, listed in Table 2, were measured and analyzed using Opticon (MJ Research). Amplification conditions were as follows: 5 s at 95°C, 20 s at 60°C, and 15 s at 72°C for 49 cycles. G3pdh was used as the internal control.

Table 2. List of PCR primers.

Genes Sequences Product size (bp)
Mmp-9: forward 5’ -GCCCTGGAACTCACACGACA-3’ 85
reverse 5’-TTGGAAACTCACACGCCAGAAG-3’
Mmp-2: forward 5’-CTCCTACAACAGCTGTACCAC-3’ 182
reverse 5’-CATACTTGTTGCCCAGGAAAG-3’
Tgf-β1: forward 5’- ATCGACATGGAGCTGGTGAAA-3’ 76
reverse 5’- TGGCGAGCCTTAGTTTGGA-3’
G3pdh: forward 5’- ACCACAGTCCATGCCATCAC-3’ 452
reverse 5’- TCCACCACCCTGTTGCTGTA-3’
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Serum cholesterol measurement

Concentrations of serum cholesterol were measured in ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice on days 0, 7, and 28 after initiation of AngII infusion. Measurements were performed by JaICA, Nikken Seil Co., Ltd (Shizuoka, Japan).

Statistical analysis

Non-parametric analyses, including the Mann-Whitney U-test or Kruskal-Wallis test with Scheffe’s and Steel-Dwass post hoc analyses, and ordinal logistic regression analyses were conducted using Ekuseru-Toukei 2012 software (Social Survey Research Information Co., Ltd.). Data are expressed as mean ± standard deviation (SD). P<0.05 was considered statistically significant.

Supporting information

S1 File

(PDF)

Click here for additional data file. (2.1MB, pdf)

Acknowledgments

We thank Mr. Masayoshi Takatani and other members in the Radiation Research Center for Frontier Science, Institute for Radiation Biology and Medicine at Hiroshima University for their technical assistances and Dr. Kenichi Satoh in the Center for Data Science Education and Research at Shiga University for valuable advice in the statistical analysis. We also appreciate for all member the Natural Science Center for Basic Research and Development (N-BARD) at Hiroshima University.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

Our study was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science, and Technology (18K07878) to MY.

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Decision Letter 0

Helena Kuivaniemi

26 Mar 2020

PONE-D-20-04332

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation

PLOS ONE

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Reviewer #1: Main summary

In the paper by Bumdelger et al, the authors evaluate the role of aneurysm/dissection in the Apoe-/- osteoprotegerin (Opg)-knockout mouse model. Interestingly, the authors introduce that using the calcium-chloride model, that deficiency of Opg had larger aneurysms and increased expression of matrix metalloproteinase-9 (MMP9) and TNF-related apoptosis-inducing ligand (TRAIL). Within this paper, using 7-month-old mice, AngII-infusion into Apoe-/- Opg-/- male mice resulted in lower external abdominal aortic diameter and lower incidence of aneurysms and dissections. The group goes on to do histological analysis and finds that collagen I expression is higher in the Apoe-/- Opg-/- mice under AngII-infusion, however this did not occur under vehicle conditions (water-infused, Supplemental Fig. 3). The group looks at alpha-smooth muscle actin, vimentin, TRAIL, F4/80, and collagen I and find that within mice that experience an aneurysm, Apoe-/- Opg-/- have increased expression of actin, vimentin, TRAIL, and collagen I that results in fibrotic remodeling that may help to lower the possibility of developing a dissection or aortic rupture. While the studies are interesting, there are some details that need to be addressed for these experiments.

Major concerns

1. If I understand correctly, both with calcium-chloride and the AngII-infusion model, TRAIL levels go up significantly. The authors state that TRAIL goes up, because the decoy receptor (Opg) is no longer expressed. If this is the case, then immunostaining for TRAIL should also be increased in the water-infused mice. If TRAIL expression is only tied to Opg, then the reader would benefit from knowing this in the water-infused mice. This could be placed in the supplement and help with the overall understanding of TRAIL within these models.

2. The authors do a good job of separating out non-aneurysm from aneurysm and dissection. In Figures 1-3, the authors examine all disease types, however in Figure 4, only aneurysm tissue is examined. Is it possible to look at non-aneurysm and dissection tissues for alpha-actin, vimentin, F4/80 and collagen I? It might help to keep it all consistent from figure to figure. There was also quantification for Figure 3, but none for Figure 4. Is it possible to do this for only 1 of the 4 proteins observed? Maybe vimentin or smooth muscle actin since quantification has already been done for collagen I.

3. Have the authors evaluated collagen IV levels in the aortic basal lamina? Is it increased in the Opg-knockout similar to collagen I?

4. It might be interesting to look at MMP2 and MMP9 levels in these Opg-knockout mice. My guess is that this might be lower in the Opg-knockout mouse, but if there is excessive remodeling off the tissue, then maybe not? This could be explored at the mRNA level if needed. Methods indicate that mRNA was collected, however I could not find any data concerning this.

Minor concerns

1. Was any ultrasound data collected? If it was collected, then placing this within the manuscript would benefit the reader.

2. Methods state that the mice were aged out to 6 months, however abstract indicates 7 months. Which is correct?

3. Suprarenal aorta (SRA) appears in the Introduction first and should be defined there.

4. “Revealed” is misspelled in the Introduction.

5. Statistical analysis should be done on the incidence data in Figure 1.

6. Did the authors measure any plasma/serum measures in the mice. Since these mice would be hypercholesterolemic, did the authors look at serum cholesterol levels?

7. Did the authors look at TGF-beta or FGF in the aorta? Since the authors conclude that fibrosis remodeling has occurred in the Opg-knockout, then measurement of these factors could help to enhance the overall conclusion.

8. There doesn’t seem to be any rupture information within these studies. Can the authors report on how many mice were lost due to aortic rupture in the wildtype and knockout mice?

Reviewer #2: The paper entitled "Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation" by Bumdelger et al examined the effect of osteoprotegerin (Opg) in abdominal aneurysm. While there is already conflicting data published on the role of Opg on aneurysm: one by the same authors concluding preventive role of Opg in CaCl2-induced AAA and another by Moran et al 2014 concluding disease promoting role of Opg in AngII-induced AAA; this paper attempts to confirm the role of Opg in AngII-induced mouse model of AAA. However, at current state of the paper, many conclusions are not fully supported by the data provided. I have following concerns:

1) Figure 1: The title of Figure 1 and conclusion "Opg deficiency limits AngII-induced aortic dissection and dilatation" is overstated. External diameter is not significantly reduced with Opg deficiency. How do the authors define dissection? The authors need to classify aneurysm based on Daugherty's classification (PMID: 11606327). Were they abdominal or thoracic dissection? The data is not statistically analyzed to say preventive effect of Opg deficiency on aortic dissection. What about the mortality rates and mortality data in these experimental mice?

2) Figure 2: I could not understand the mechanism behind adventitial thickening and aortic dissection. Do the authors have any speculation on how does the adventitial thickening leads to smaller aortic diameter and dissection in ApoE-/-Opg-/- mice?

3) Figure 3: What about collagen III? Picrosirius red staining needs to be performed for differential staining of collagen I vs collagen III.

4) Figure 4. The authors show that cells accumulated in adventitial region of Apoe-/-Opg-/- mice are likely myofibroblasts since they co-express SMA and vimentin. However, the Figure 4I shows more infiltration of F4/80+ macrophages in Apoe-/-Opg-/- compared to Apoe-/- alone in 4F. And it is surprising that there are fewer macrophages and less adventitial thickening in Apoe-/- mice in 4F and 4E. How does increased Trail expression, increased inflammation, myofibroblast accumulation and fibrotic remodeling can be protective to aortic dilation and dissection? The data presented and conclusion do not match. Instead, these data imply that there would be more disease in Apoe-/-Opg-/- mice because of more inflammation.

5) Why 6-months (24 weeks) old mice are used for the aneurysm studies? Aneurysm studies are best performed in 8-12 weeks old mice.

6) Poor quality of writing: There are several grammatical, typo and spelling errors in the sentences. The paper needs significant English editing.

Reviewer #3: The paper entitled " Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation" by Kokubo et al concluded that fibrotic remodeling of the aorta induced by myofibroblast accumulation might be an important pathological event which limits AngII-induced aortic dilatation in ApoE -/- Opg -/- mice.

The conclusion of the manuscript is not substantially based upon the experimental data and is highly speculative.

Specifically:

1. The statement ‘Opg deficiency limits AngII-induced aortic dissection and dilatation’ is an overstatement of the data. The AAA definition is missing and also lacks statistical analysis.

2. No evidence to show the specificity of antibodies to detect vSMCs and fibroblasts. Single IHC may not be sufficient to draw the conclusions.

3. The interpretation that ‘Opg deficiency was found to limit AngII-induced aortic dissection and dilatation’ is not convincing.

4. There are no reports/data to show that fibrotic remodeling of the aorta might protects against AAA.

5. Why the authors used 6 month old mice?

6. Since aortic dissection in these mice occur at early stage of the disease (4-10 days), determination of visible blood at day 28 may not be a good indicator for aortic dissection.

7. Please check for grammatical errors.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Chetan P Hans

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PLoS One. 2020 Jul 2;15(7):e0235553. doi: 10.1371/journal.pone.0235553.r002

Author response to Decision Letter 0

10 May 2020

Re: PONE-D-20-0433

We thank the Reviewers for the helpful and constructive comments on our manuscript, entitled “Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation.” We have considered the Reviewers’ comments carefully and have revised the manuscript to address their concerns and questions. Our responses to the Reviewers’ comments are detailed point-by-point below.

We hope that all concerns have been adequately addressed and that the revised manuscript is now suitable for publication in PLOS ONE. Please do not hesitate to contact us if you require further information.

Reviewer #1: Main summary

In the paper by Bumdelger et al, the authors evaluate the role of aneurysm/dissection in the Apoe-/- osteoprotegerin (Opg)-knockout mouse model. Interestingly, the authors introduce that using the calcium-chloride model, that deficiency of Opg had larger aneurysms and increased expression of matrix metalloproteinase-9 (MMP9) and TNF-related apoptosis-inducing ligand (TRAIL). Within this paper, using 7-month-old mice, AngII-infusion into Apoe-/- Opg-/- male mice resulted in lower external abdominal aortic diameter and lower incidence of aneurysms and dissections. The group goes on to do histological analysis and finds that collagen I expression is higher in the Apoe-/- Opg-/- mice under AngII-infusion, however this did not occur under vehicle conditions (water-infused, Supplemental Fig. 3). The group looks at alpha-smooth muscle actin, vimentin, TRAIL, F4/80, and collagen I and find that within mice that experience an aneurysm, Apoe-/- Opg-/- have increased expression of actin, vimentin, TRAIL, and collagen I that results in fibrotic remodeling that may help to lower the possibility of developing a dissection or aortic rupture. While the studies are interesting, there are some details that need to be addressed for these experiments.

Major concerns

1. If I understand correctly, both with calcium-chloride and the AngII-infusion model, TRAIL levels go up significantly. The authors state that TRAIL goes up, because the decoy receptor (Opg) is no longer expressed. If this is the case, then immunostaining for TRAIL should also be increased in the water-infused mice. If TRAIL expression is only tied to Opg, then the reader would benefit from knowing this in the water-infused mice. This could be placed in the supplement and help with the overall understanding of TRAIL within these models.

We thank the Reviewer for the suggestion. We did not detect significant differences in Trail expression between H2O-infused ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice. However, AngII infusion significantly increased Trail expression only in ApoE-/-Opg-/- mice. These data were added to Supplemental Figures 5D and 5F.

2. The authors do a good job of separating out non-aneurysm from aneurysm and dissection. In Figures 1-3, the authors examine all disease types, however in Figure 4, only aneurysm tissue is examined. Is it possible to look at non-aneurysm and dissection tissues for alpha-actin, vimentin, F4/80 and collagen I? It might help to keep it all consistent from figure to figure. There was also quantification for Figure 3, but none for Figure 4. Is it possible to do this for only 1 of the 4 proteins observed? Maybe vimentin or smooth muscle actin since quantification has already been done for collagen I.

We thank the Reviewer for the suggestion. As suggested, we performed IHC studies using antibodies against α-SMA, vimentin, and F4/80 for all disease types and also quantified the α-SMA expressing area in the adventitia. These data were added to Figure 4 (former Figure 4 has been renumbered to be Figure 5). Corresponding changes were also made to the manuscript (page 7, lines 18-19).

3. Have the authors evaluated collagen IV levels in the aortic basal lamina? Is it increased in the Opg-knockout similar to collagen I?

Unfortunately, we did not perform that experiment. We did, however, perform IHC studies using an anti-collagen III antibody and found that collagen III was not expressed in the vascular wall of both ApoE-/-Opg+/+ and ApoE-/-Opg-/- mice. These data were added to Supplemental Figure 4B.

4. It might be interesting to look at MMP2 and MMP9 levels in these Opg-knockout mice. My guess is that this might be lower in the Opg-knockout mouse, but if there is excessive remodeling off the tissue, then maybe not? This could be explored at the mRNA level if needed. Methods indicate that mRNA was collected, however I could not find any data concerning this.

We performed quantitative RT-PCR to measure Mmp2 and Mmp9 transcript levels. No significant differences were detected in Mmp9 levels between ApoE-/- Opg+/+ and ApoE-/- Opg-/- mice, although Mmp2 levels were elevated in ApoE-/- Opg-/- mice. Interestingly, AngII infusion did not influence Mmp2 levels, implying that either Mmp2 or Mmp9 is not involved in the histological changes induced by AngII infusion. These data were included in Supplemental Figures 5A-5C.

Minor concerns

1. Was any ultrasound data collected? If it was collected, then placing this within the manuscript would benefit the reader.

We appreciate the Reviewer’s suggestion. However, we unfortunately did not collect ultrasound data.

2. Methods state that the mice were aged out to 6 months, however abstract indicates 7 months. Which is correct?

We thank the Reviewer for pointing this out. The manuscript was revised to clarify that AngII infusions started when mice were 6 months of age. Mice were sacrificed at 7 months of age.

3. Suprarenal aorta (SRA) appears in the Introduction first and should be defined there.

Suprarenal aorta (SRA) is defined in the manuscript on page 5 (line 5). We also added a representative image of the aorta to help explain the portion to which the SRA corresponds (Supplemental Figure 1).

4. “Revealed” is misspelled in the Introduction.

This typographical error was corrected, accordingly (page 4, line 9).

5. Statistical analysis should be done on the incidence data in Figure 1.

Statistical analysis was performed for Figure 1C. We used ordinal logistic regression analysis.

6. Did the authors measure any plasma/serum measures in the mice. Since these mice would be hypercholesterolemic, did the authors look at serum cholesterol levels?

Cholesterol concentrations were available and added to Supplemental Figure 2G.

7. Did the authors look at TGF-beta or FGF in the aorta? Since the authors conclude that fibrosis remodeling has occurred in the Opg-knockout, then measurement of these factors could help to enhance the overall conclusion.

We thank the Reviewer for the helpful suggestion, and agree that Tgf-β may have an effect on fibrosis remodeling. We measured Tgf-β1 levels by RT-PCR and found that they increased after AngII infusion, consistent with a role for Tgf-β1 in enhancing fibrosis remodeling. We added an explanation of this to the manuscript (page 7, lines 10-13), and included the data in Figure 3C.

8. There doesn’t seem to be any rupture information within these studies. Can the authors report on how many mice were lost due to aortic rupture in the wildtype and knockout mice?

Unfortunately, we only counted the number of dead mice and did not confirm whether their deaths were due to aortic rupture. Supplemental Figure 2F provides the survival data.

 

Reviewer #2: The paper entitled "Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation" by Bumdelger et al examined the effect of osteoprotegerin (Opg) in abdominal aneurysm. While there is already conflicting data published on the role of Opg on aneurysm: one by the same authors concluding preventive role of Opg in CaCl2-induced AAA and another by Moran et al 2014 concluding disease promoting role of Opg in AngII-induced AAA; this paper attempts to confirm the role of Opg in AngII-induced mouse model of AAA. However, at current state of the paper, many conclusions are not fully supported by the data provided. I have following concerns:

1) Figure 1: The title of Figure 1 and conclusion "Opg deficiency limits AngII-induced aortic dissection and dilatation" is overstated. External diameter is not significantly reduced with Opg deficiency. How do the authors define dissection? The authors need to classify aneurysm based on Daugherty's classification (PMID: 11606327). Were they abdominal or thoracic dissection? The data is not statistically analyzed to say preventive effect of Opg deficiency on aortic dissection. What about the mortality rates and mortality data in these experimental mice?

We thank the Reviewer for these insightful comments. As suggested, we revised the title and first paragraph of the Results section to be more commensurate in scope with the data.

We did not initially follow Daugherty’s classification, since Moran’s group did not use that classification system. Nonetheless, we newly evaluated aneurysms based on the presence or absence of hematoma by morphological observation, similar to Daugherty’s classification. We cite Daugherty’s article and clarify that our classification method is a modified version of Daugherty’s method (page 5, lines 14-17). Our focus was mainly abdominal dissection at the SRA, although dissection was also observed in the thoracic region.

As mentioned by Reviewer #1, we statistically analyzed the incidence rate of three disease phases using ordinal logistic regression. In result, there was a non significant tendency for aneurysm and aortic dissection in ApoE-/- Opg-/- mice. We included the mortality data in Supplemental Figure 2F.

2) Figure 2: I could not understand the mechanism behind adventitial thickening and aortic dissection. Do the authors have any speculation on how does the adventitial thickening leads to smaller aortic diameter and dissection in ApoE-/-Opg-/- mice?

To address this comment, we provide a schematic and description of how we believe adventitial thickening leads to smaller aortic diameter and dissection in ApoE-/-Opg-/- mice (Supplemental Figure 6).

We speculate that administration of AngII in the ApoE-KO mouse model leads to inflammation, destruction of aortic tissue, and expression of inflammatory cytokines. In Opg deficient mice, AngII induces the expression of Trail, which may stimulate the appearance, proliferation, and migration of myofibroblasts, as indicated in previous reports. This accumulation of myofibroblasts may result in adventitial thickening with fibrosis, e.g., as a result of collagen I deposition. A substantial amount of collagen I in the adventitia could suppress dilatation of the inner diameter of the suprarenal aorta (SRA) by maintaining the stiffness and structural integrity of the lumen (Dobrin PB, et al. PMID: 7953454, Malfait F. PMID: 29709596). Thus, the characteristic changes in the adventitia of ApoE-/-Opg-/- mice, including the accumulation of myofibroblasts and collagen I, may underlie the suppression of aneurysm formation and obscure the influence of tissue destruction. Supporting this potential mechanism is a study by Dobrin et al., which found in ex vivo experiments that additional collagenase, rather than elastase, led to the enlargement of the lumen of blood vessels (Dobrin PB, et al. PMID: 7953454).

3) Figure 3: What about collagen III? Picrosirius red staining needs to be performed for differential staining of collagen I vs collagen III.

As suggested, we performed IHC experiments with antibodies against collagen type III. The expression of collagen III was much lower than that of collagen I and was not upregulated in response to AngII stimulation. We confirmed that the anti-collagen III antibody works by using a section of skin tissue as a positive control (Supplemental Figure 4B).

4) Figure 4. The authors show that cells accumulated in adventitial region of Apoe-/-Opg-/- mice are likely myofibroblasts since they co-express SMA and vimentin. However, the Figure 4I shows more infiltration of F4/80+ macrophages in Apoe-/-Opg-/- compared to Apoe-/- alone in 4F. And it is surprising that there are fewer macrophages and less adventitial thickening in Apoe-/- mice in 4F and 4E. How does increased Trail expression, increased inflammation, myofibroblast accumulation and fibrotic remodeling can be protective to aortic dilation and dissection? The data presented and conclusion do not match. Instead, these data imply that there would be more disease in Apoe-/-Opg-/- mice because of more inflammation.

We describe the mechanism by which we speculate Trail expression is increased, inflammation is stimulated, myofibroblasts accumulate, and how fibrosis could suppress aortic dilatation and dissection in Supplemental Figure 6.

In further experiments, we detected an increase in macrophages, but no upregulation of Mmps, suggesting that inflammation does not play a major role in the observed histological changes. On the other hand, the thickened adventitia was filled with myofibroblasts and had substantial collagen I accumulation. As mentioned above in response to point #2, the presence of collagenase, rather than elastase, has been shown to cause enlargement of the lumen of blood vessels in a previous ex vivo study (Dobrin PB, et al. PMID: 7953454). These data indicate that fibrotic remodeling can be protective against aortic dilatation and dissection.

5) Why 6-months (24 weeks) old mice are used for the aneurysm studies? Aneurysm studies are best performed in 8-12 weeks old mice.

Our initial goal was to reproduce the experiments performed by Moran’s group, and thus matched the ages of mice with their study.

6) Poor quality of writing: There are several grammatical, typo and spelling errors in the sentences. The paper needs significant English editing.

We had the manuscript checked and edited by a native English speaker.

 

Reviewer #3: The paper entitled " Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation" by Kokubo et al concluded that fibrotic remodeling of the aorta induced by myofibroblast accumulation might be an important pathological event which limits AngII-induced aortic dilatation in ApoE -/- Opg -/- mice.

The conclusion of the manuscript is not substantially based upon the experimental data and is highly speculative.

Specifically:

1. The statement ‘Opg deficiency limits AngII-induced aortic dissection and dilatation’ is an overstatement of the data. The AAA definition is missing and also lacks statistical analysis.

We thank the Reviewer for raising this point. We agree and have revised the manuscript to avoid overstating the present findings (page 5, line 2, and title) and to include the definition of AAA (page 13, lines 17-19).

2. No evidence to show the specificity of antibodies to detect vSMCs and fibroblasts. Single IHC may not be sufficient to draw the conclusions.

In the previous version of the manuscript, myofibroblasts were identified by IHC using anti-α-SMA or anti-vimentin antibodies. However, we agree that single IHC data cannot be used definitively to identify myofibroblasts. Accordingly, we performed immunofluorescence-based double staining with anti-α-SMA and anti-vimentin antibodies. Cells which were positive for both markers were considered to be myofibroblasts, and cells positive only for α-SMA were considered to be vSMCs (Skalli O, et al. PMID: 2918221).

3. The interpretation that ‘Opg deficiency was found to limit AngII-induced aortic dissection and dilatation’ is not convincing.

We added a discussion of how we arrived at that interpretation in Supplemental Figure 6 (and its accompanying legend). As mentioned in our reply to a similar comment from Reviewer #2, we believe that adventitial thickening accompanying collagen I accumulation has a preventive effect on enlargement of the aortic lumen and total outer diameter. According to a previous study, it was found in ex vivo experiments that additional collagenase, rather than elastase, led to the enlargement of the lumen of blood vessels (Dobrin PB, et al. PMID: 7953454). When this finding is applied to the present context, the accumulation of collagen I could be one of the causes underlying the preventive effect on enlargement of the aortic lumen.

4. There are no reports/data to show that fibrotic remodeling of the aorta might protects against AAA.

We agree with the Reviewer that there is no publication directly on point. However, as mentioned in the reply to the previous comment, Dobrin et al. found that a reduction in collagen I decreases the stiffness of the vascular wall (Dobrin PB, et al. PMID: 7953454). Moreover, according to a study by Malfait et al., corruption of collagen promoted the formation of aneurysms and dissection (Malfait F. et al. PMID: 29709596). Taken together, we believe our speculation that collagen I accumulation in the adventitia results in increased stiffness of the vascular wall to be reasonable.

5. Why the authors used 6 month old mice?

Our initial goal was to reproduce the experiments conducted by Moran’s group. Accordingly, we used the experimental conditions described in their paper, including the age of mice.

6. Since aortic dissection in these mice occur at early stage of the disease (4-10 days), determination of visible blood at day 28 may not be a good indicator for aortic dissection.

As mentioned above, our initial goal was to reproduce the experiments conducted by Moran’s group. To this end, we mirrored the conditions they used, including those for observation period (i.e., 28 days after AngII infusion). Accordingly, we did not perform earlier stage observations. Classification of aneurysms by Daugherty’s group was also performed 28 days after AngII infusion, which is when the severity of aneurysms shows diversity in this AAA model (Daugherty A, et al. PMID: 11606327). Based on the above, we believe 28 days to be appropriate for observing the phenotype of mice.

7. Please check for grammatical errors.

We had the manuscript checked and edited by a native English speaker.

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Decision Letter 1

Helena Kuivaniemi

8 Jun 2020

PONE-D-20-04332R1

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation

PLOS ONE

Dear Dr. Kokubo,

Thank you for submitting your manuscript to PLOS ONE. After careful review, we consider it to have merit but it does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Please respond to all the comments and provide additional information on antibodies and primers used in your study.

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We look forward to receiving your revised manuscript.

Kind regards,

Helena Kuivaniemi, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (if provided):

Please provide tables in the supplementary material with details on the antibodies (vendor, cat#, lot#, specificity , literature citation using the same antibody) and primers (sequence, annealing temperature, PCR product size) used for the study

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

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Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please make the following small editorial changes.

1. Hematoma is misspelled on page 13, second to last line.

2. Catalog numbers for all antibodies should be included to help with replication of studies.

3. Need a hyphen for alpha-SMA in Figure 4 legend

4. Need MMP2 primer sequence in the Methods section.

5. Should state both "MMP-2 and MMP-9" in Discussion section, page 10, second to last line.

Reviewer #2: All the comments have been nicely addressed by the authors with addition of data. I have no further questions.

Reviewer #3: All the comments have been addressed adequately by the authors. The reviewer has no more major concerns.

**********

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Chetan P Hans

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PLoS One. 2020 Jul 2;15(7):e0235553. doi: 10.1371/journal.pone.0235553.r004

Author response to Decision Letter 1

16 Jun 2020

Re: PONE-D-20-04332R1

Additional Editor Comments (if provided):

Please provide tables in the supplementary material with details on the antibodies (vendor, cat#, lot#, specificity , literature citation using the same antibody) and primers (sequence, annealing temperature, PCR product size) used for the study

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

Reviewer #2: All comments have been addressed

Reviewer #3: All comments have been addressed

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: N/A

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Please make the following small editorial changes.

1. Hematoma is misspelled on page 13, second to last line.

This typographical error was corrected accordingly.

2. Catalog numbers for all antibodies should be included to help with replication of studies.

We added a table in the manuscript to show catalog numbers for all antibodies followed by reviewer’s suggestion. We also changed text to cite this table in the section fo materials and methods, pp15 line 5-6.

3. Need a hyphen for alpha-SMA in Figure 4 legend

We deleted α in the Figure 4 legend (page 23) because we have defined the α-smooth muscle actin as SMA in page 4, line 14.

4. Need MMP2 primer sequence in the Methods section.

The MMP2 primer sequence has already been added in the method section (page 15, last line). To avoid confusion, we added an additional table in the manuscript to provide details on primers we used. We also changed text to cite this table in the section fo materials and methods, pp15 line 15.

5. Should state both "MMP-2 and MMP-9" in Discussion section, page 10, second to last line.

As followed reviewer’s suggestion, we stated both MMP-9 and MMP-2.

Reviewer #2: All the comments have been nicely addressed by the authors with addition of data. I have no further questions.

Reviewer #3: All the comments have been addressed adequately by the authors. The reviewer has no more major concerns.

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: Yes: Chetan P Hans

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Decision Letter 2

Helena Kuivaniemi

18 Jun 2020

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation

PONE-D-20-04332R2

Dear Dr. Kokubo,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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kind regards,

Helena Kuivaniemi, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Acceptance letter

Helena Kuivaniemi

22 Jun 2020

PONE-D-20-04332R2

Disruption of Osteoprotegerin has complex effects on medial destruction and adventitial fibrosis during mouse abdominal aortic aneurysm formation

Dear Dr. Kokubo:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Professor Helena Kuivaniemi

Academic Editor

PLOS ONE

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