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. 2021 Mar 4;16(3):e0235454. doi: 10.1371/journal.pone.0235454

Entelon (vitis vinifera seed extract) reduces degenerative changes in bovine pericardium valve leaflet in a dog intravascular implant model

Gab-Chol Choi 1,2,‡, Sokho Kim 3,‡, Md Mahbubur Rahman 3, Ji Hyun Oh 4, Yun Seok Cho 4, Hong Ju Shin 4,*
Editor: Andreas Zirlik5
PMCID: PMC7932063  PMID: 33661896

Abstract

Background and aims

Inflammation and calcification are major factors responsible for degeneration of bioprosthetic valve and other substitute heart valve implantations. The objective of this study was to evaluate the anti-inflammatory and anti-calcification effects of Entelon150® (consisting of grape-seed extract) in a beagle dog model of intravascular bovine pericardium implantation.

Methods

In total, 8 healthy male beagle dogs were implanted with a bovine pericardium bilaterally in the external jugular veins and divided into two groups. Animals in the Entelon150® group (n = 4) were treated with 150 mg of Entelon150® twice daily for six weeks after surgery. The negative control (NC) group (n = 4) was treated with 5 ml of saline using the same method. After six weeks, we measured the calcium content, performed histological examination, and performed molecular analysis.

Results

The calcium content of implanted tissue in the Entelon150® group (0.56±0.14 mg/g) was significantly lower than that in the NC group (1.48±0.57 mg/g) (p < 0.05). Histopathological examination showed that infiltration of chronic inflammatory cells, such as fibroblasts and macrophages, occurred around the graft in all groups; however, the inflammation level of the implanted tissue in the Entelon150® group was s lower than that in the NC group. Both immunohistochemical and western blot analyses revealed that bone morphogenetic protein 2 expression was significantly attenuated in the Entelon150® group.

Conclusions

Our results indicate that Entelon150® significantly attenuates post-implantation inflammation and degenerative calcification of the bovine pericardium in dogs. Therefore, Entelon150® may increase the longevity of the bovine pericardium after intravascular implantation.

Introduction

Bovine pericardium is widely used in patch material during vascular surgery. It is also used in bioprosthetic heart valve leaflets with specific treatment to increase its longevity. Heart valves made of a bovine pericardium are safe, offer improved hemodynamics, have less risk of thrombosis, and do not need long-term anticoagulant therapy [1, 2]. However, the durability of the bovine pericardium is a major problem, as it is prone to valve calcification, structural deterioration, and eventual failure. Several approaches to reduce calcification have been attempted, including systemic anti-calcification agent administration. However, many of these approaches have been either ineffective or have produced unwanted side effects [3]. To overcome this drawback, studies on the complementary treatment of calcification have focused on traditional herbal medicines recently [4–6].

Grape fruit (Vitis vinifera) is one of the most important and popular fruit crops worldwide because of its high phytochemical content and its nutritional value. All parts of this fruit have been used as dietary supplements to treat or prevent various diseases [7–9]. In general, grape fruit is rich in phenols, flavonoids, and fatty acids. The anti-inflammatory [7, 9] and anti-oxidative [7] effects of grape seed extract (GSE) are now well established.

Interleukin-6 (IL-6) plays an important role in increasing bone morphogenic protein 2/4 (BMP-2/4) expression in vessels and valve tissue, thereby leading to vascular calcification [3]. It has been shown that application of GSE reduces IL-6 activity in different disease models [10, 11].

To the best of our knowledge, there have been no studies examining the effects of GSE on bovine pericardium implants. It is thus unknown whether GSE could prevent bovine pericardium calcification and degeneration. Therefore, the purpose of this study was to investigate the anti-inflammatory and anti-calcification effects of Entelon150® on intravenous bovine pericardium implants in beagle dogs.

Methods

Animal and experimental design

Eight healthy male beagle dogs (20 weeks old; mean body weight 9.76 ± 0.32 kg, range 8.00–10.80 kg) were used in this study which were purchased from ORIENT BIO Inc. (Seongnam, Gyeonggi-do, Republic of Korea). They were housed separately in stainless steel cages (W 895 × L 795 × H 765 mm) in an environmentally controlled room (temperature 23 ± 3°C, relative humidity 55 ± 15%, ventilation frequency 10–20 times/hr, light cycle 8 am to 8 pm, illumination 150 to 300 Lux). Food and sterilized water were available ad libitum. Animals were divided equally into two groups: 1) Negative control (NC) group: vehicle-treated after prosthetic implantation; 2) Entelon150®-treated group: treated with grape seed extract (Entelon150®; Lot number: RNR601, Hanlim Pharm. Co., Ltd. Yongin-si, Korea), 150 mg per animal twice daily for six weeks after implantation. The mouth of the animal was opened to its natural position in the breeding box, and the test article was placed on the tongue. The animal’s mouth was then shut, and the neck was gently stroked until the animal swallowed. All animals were closely monitored during the experimental period, and we observed no clinical symptoms. This study was approved by the Institutional Animal Care and Use Committee at the KNOTUS Co., Ltd., Incheon-si, Korea (Certificate No: IACUC 19-KE-132).

Surgical procedure for implantation and postoperative care

Each animal was anesthetized with an intravenous injection of Zoletil 50 (VIRBAC, France; 5 mg/kg) and xylazine (Rompun®, Bayer AG, Germany; 2.5 mg/kg). After intubation with a 3.0-mm endotracheal tube, inhaled isoflurane was used to maintain anesthesia. All animals received 0.9% saline (10 ml/kg/h) intravenously throughout the surgical procedure. Intravenous antibiotic cephradine (30 mg/kg) and intravenous analgesic tramadol (2 mg/kg) were injected preoperatively as described previously [12]. The skin was incised along the ventral cervical midline, and blunt separation was performed to expose the left external jugular vein from the sternohyoid muscle. After administration of heparin (50 IU/kg, IV), the left jugular vein was temporarily blocked using 4–0 silk and 5 French feeding tubes. An 8-mm longitudinal incision was then made in the jugular vein using a no. 11 surgical blade. A commercially available bovine pericardium (PERIBORN® Bovine Pericardium, BP0506, Taewoong Medical Co., Ltd. Gimpo-si, South Korea) was used in this study. Before implantation, the Bovine pericardium was rinsed for 30 min in 500 ml of sterile physiological saline. An approximately 3-mm rectangular bioprosthetic was fixed to the inner wall of the jugular vein using a 6–0 polypropylene running suture. The jugular vein was closed by angioplasty using a 6–0 polypropylene running suture. The bovine pericardium was also implanted into the right external jugular vein as described above. The schematic diagram below shows the anatomy of the jugular vein in beagle dogs, relevant to this surgical procedure (Fig 1). Postoperatively, intramuscular enrofloxacine (10 mg/kg, bid), intravenous cephradine (30 mg/kg bid or tid), intravenous tramadol (2–3 mg/kg bid or tid) and intravenous cimetidine (10 mg/kg, bid) were performed for three days [12]. Respiratory rate was monitored by the movement of the abdominal and chest wall (breath/min) twice daily. Heart rate and ECG was recorded with a Cardiofax ECG-9020 electrocardiograph (Nihon Kohden, Tokyo, Japan) once daily for three days [13]. Beside these normal clinical signs were closely monitored daily until end of the experiment to identify any abnormality due to surgery or any side effect for administering Entelon150 including changes of body weight, feeding and drinking behavior, urination and defecation frequency, bleeding, salivating, vomiting, abnormality or redness of skin and eye color, abnormal sound and abnormal movement but none were observed in entire experimental period. Body weight was measured just before starting experiment and then once a week until six weeks (at o, 1, 2, 3, 4, 5 and 6 weeks) (Fig 2).

Fig 1. Anatomical diagram of a beagle’s jugular vein during the implantation procedure.

Fig 1

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Yellow star indicates the incision site of jugular vein.

Fig 2. Effects of bioprosthetic valve implantation and Entelon® treatment on body weight and vascular patency as measured by CT-Scan.

Fig 2

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Arrow indicates the incision and implanted site in jugular vein.

Measurement of vascular patency

Vascular patency was checked using the color doppler mode of an ultrasonic device (LOGIQ e Ultrasound; GE Healthcare, Fairfield, USA) 6 weeks after the bioprosthetic implantation. Angiography was also performed with a bolus intravenous injection of 2 ml/kg iohexol (Omnipaque™, 300 mg I/ml; GE Healthcare) using a CT-scanner (CT; 16-channel multidetector; BrightSpeed Elite, GE Healthcare, Fairfield, CT, USA) for additional confirmation just after ultrasonography and immediately before sacrifice at 6 weeks.

Sample collection

Six weeks after surgery and immediately after measuring vascular patency, the animals were euthanized by intravenous Propofol (10 mg/kg) and then intravenous Pentobarbital (50 mg/kg). and both the left and right external jugular veins were removed. Only implanted prosthetic was collected from the right external jugular vein and connective tissues around the implanted prosthetic was carefully removed and then stored in a cryogenic freezer maintained at about -80°C until calcium quantification. However, half of the left external jugular vein along with connective tissue was fixed in a 10% neutral buffered formalin solution for histopathological examination, and from the other half part only prosthetic was stored in a cryogenic freezer maintained at -80°C for western blotting.

Measurement of calcium content

Moisture was removed from the stored implant through 24 h of freeze-drying. After weighing the implants, they were suspended in a beaker containing 5 ml of aqua regia solution. Hydrogen peroxide (H2O2; 5 ml) was then added to the beaker, and the beaker was slowly heated to 70–80°C on a hot plate for 6 h to dissolve the tissue. The solution was then heated for a further 5 h at 140–150°C. An additional 5 ml of H2O2 was then added. Subsequently, each sample was diluted to a total volume of 50 ml with distilled water. The diluted samples were used to measure the calcium content.

Histopathological examination

For histopathological examination, each sample was soaked in 10% neutralized buffered formalin and processed using standard methods. Paraffin-embedded tissue was sliced into 4-μm-thick sections and stained with hematoxylin and eosin (H & E). For H & E-stained slides, infiltration of inflammatory cells around the implanted bovine pericardium was quantified according to the following criteria: 0: none, 1: weak inflammatory cell infiltration, 2: moderate inflammatory cell infiltration, 3: severe inflammatory cell infiltration.

For immunohistochemical analysis, the sections were deparaffinized following standard protocols. The sections were then incubated in 3% hydrogen peroxide for 10 min to inactivate endogenous peroxidase and then blocked by incubating the sections for 1 h in 5% bovine serum albumin. The sections were then incubated overnight with anti-alpha smooth muscle actin (α-SMA) (1:1600, ab5964, Abcam) and bone morphogenetic protein 2 (BMP-2) (1:200, orb251474, Biorbyt) primary antibodies at 4°C. The sections were washed and incubated with secondary anti-rabbit (α-SMA) or anti-mouse (BMP-2) antibodies for 30 min at approximately 20°C. The sections were then washed, counterstained with Mayers Hematoxylin, and mounted on slides. The percentage of stained area (%) was compared between groups. For immunohistochemically stained slides, the expression area was analyzed using a slide scanner (Axio Scan Z1, Carl Zeiss, Germany), and the slides were then subjected to image analysis (ZEN, Carl Zeiss, Germany).

Western blotting

Cryogenically frozen samples were homogenized using RIPA buffer, after which proteins were extracted and quantified with a protein assay kit (Bio-Rad, CA, USA). A sample for electrophoreses was prepared by quantifying the sample’s protein content, and the sample was electrophoresed on 10–14% acrylamide gel for 120 minutes. The protein was then transferred to a polyvinylidene difluoride (PVDF) membrane, and non-specific protein binding sites were removed using blocking buffer. The PVDF membrane was incubated with interleukin-6 (IL-6), osteopontin (OPN), and bone morphogenetic protein 2 (BMP-2) primary antibodies at about 4°C for 6 hours or more. After the primary antibody reaction, the membrane was exposed to the secondary antibody (diluted 1: 10,000). After the reaction was complete, the cells were washed with PBS-T buffer (0.5% Tween 20 in phosphate buffered saline) and the sample was developed using an enhanced chemiluminescence (ECL) reagent for immunoblot analysis. The color-completed sample was analyzed using an image analyzer. β-actin was used as intrinsic control. Finally, the ECL signal was quantitated using the pixel density analysis algorithm within ImageJ software (National Institute of Health, NY, USA). The relative band density was calculated as follows: relative band density = (specific band density/β-actin band density) × 100.

Statistical analysis

All data are expressed as mean ± SD. Statistical analyses were performed using GraphPad Prism 5.0 software (GraphPad Software, Inc., San Diego, CA, USA). An unpaired t-test was used to compare two groups. All statistical tests were two-sided, and significance was defined as P < 0.05.

Results

There were no differences in body weight between the control and Entelon150®-treated groups (Fig 2A). Vascular patency was evaluated using ultrasonography and CT scanning. No interruption of flow patency was observed on either CT scans (Fig 2B and 2C) or ultrasonographs (Fig 3). Although there are no occlusions visible around the vessel or in the implants in either control or Entelon150®-treated groups, the vessels shown have an overall smaller and more irregular diameter might be for suturing effect (Fig 2). The Ca2+ level in the Entelon150®-treated group (0.56±0.14 mg/g) was significantly lower than that in the NC group (1.48±0.57 mg/g) (P < 0.05, Fig 4). Western blot analysis showed that BMP-2 levels in the Entelon150®-treated group (82.21±11.20%) were significantly lower (P < 0.001) than in the NC group (100.00±4.63%). Western blot analysis also revealed that IL-6 levels in the Entelon150®-treated group (55.36±5.49%) were significantly lower (P < 0.001) than NC group (100.00±10.30%) indicating the significant attenuation of inflammation. However, the expression of OPN was not significantly different between two groups (Fig 5). Histopathological examination revealed infiltration of chronic inflammatory cells such as fibroblasts and macrophages around the graft in all groups. However, the inflammation level of the Entelon150®-treated group (1.50±0.58%) was significantly lower (P < 0.001) than the NC group (2.25±0.96%). In particular, a basophilic substance presumed to be the earliest sign of calcium deposition was observed in the NC group between the intercellular matrixes of the peri-graft tissue (Fig 6). Immunohistochemical staining revealed that BMP-2 levels in the Entelon150®-treated group (1.27±0.06%) were significantly (P < 0.05) lower than those in the NC group (1.67±0.31%). However, the expression of α-SMA was not significantly different between the two groups (Fig 7).

Fig 3. Effects of bioprosthetic valve implantation and Entelon® treatment on vascular patency as measured by ultrasonography.

Fig 3

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NC: Negative control group. Entelon®: Entelon®-treated group. The fluent vascular patency was confirmed by Ultrasonography”.

Fig 4. Therapeutic efficacy of Entelon® treatment on the calcium content in bioprosthetic valve implants.

Fig 4

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NC: Negative control group, Entelon®: Entelon®-treated group. The data are reported as mean ± SD. *: p < 0.05; and ***: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus the NC group.

Fig 5. Effects of bioprosthetic valve implantation and Entelon® treatment on the protein expression of BMP-2, IL-6 and OPN as analyzed by western blot.

Fig 5

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β-Actin was used as an internal control. NC: Negative control group, Entelon®: Entelon®-treated group. IL-6: Interleukin-6, OPN: osteopontin, BMP-2: bone morphogenetic protein 2. The data are reported as mean ± SD. *: p < 0.05; and ***: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus the NC group.

Fig 6. Evaluation of the therapeutic efficacy of Entelon® using histological images analysis.

Fig 6

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NC: Negative control group, Entelon®: Entelon®-treated group. The data are reported as mean ± SD. ***: p < 0.001, Bonferroni post hoc test following one-way ANOVA versus the NC group.

Fig 7. Evaluation of the therapeutic efficacy of Entelon® using histological images analysis.

Fig 7

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NC: Negative control group, Entelon®: Entelon®-treated group. The data are reported as mean ± SD. *: p < 0.05, Bonferroni post hoc test following one-way ANOVA versus the NC group.

Discussion

This study was the first to investigate the anti-inflammatory and anti-calcification effects of Entelon150® using a beagle dog model of intravenous bovine pericardium implantation. Chronic inflammation and calcification are major signs of the structural degeneration, dysfunction, and failure of bioprosthetic valve made of bovine pericardium [3, 14, 15]. We hypothesized that Entelon150® would reduce the expression of inflammatory cytokines in the vascular tissue of bovine pericardial implants, thereby suppressing calcification and bovine pericardium. In this study we demonstrated that Entelon150® treatment had two effects: significantly attenuating both inflammation and calcification in a beagle dog model of intravascular bovine pericardial implantation.

Calcification or degenerative changes of implant valve in human usually takes longtime [16]. However, degeneration was observed comparatively with in shorter time in experimental implant model; evaluated after 14 and 30 days [17], 4 and 12 weeks [14], 3 weeks [18], 6 weeks [3, 19, 29] implantation. Subcutaneous and intramuscular implantation models are usually performed to investigate BHV degenerative changes or calcification [14, 17–19]. Unfortunately, subcutaneous and intramuscular models are not appropriate as graft tissue experienced hemodynamic stress due to the direct blood contact or circulating factors inside the heart or great vessels. Therefore, intravascular implant model became popular model [3, 29]. We therefore performed intravascular implant model.

Indeed, immunological rejection of heterologous tissue is a challenging medical problem contributing to post-implant xenograft degeneration [19]. Our histopathological results are consistent with previous studies observing infiltration of chronic inflammatory cells such as fibroblasts and macrophages around the graft. The inflammation levels of the Entelon150®-treated group was significantly lower than the negative control group. In addition, a basophilic substance, presumed to be the earliest sign of calcium deposition, was observed between the intercellular matrixes of the peri-graft tissue in the negative control group. The basophilic staining using the H & E stain method indicated calcium deposition [20]. The results suggest that administration of Entelon150® lowered inflammation levels and inhibited calcium deposition in the tissues surrounding the graft.

In addition, we found that bovine pericardium triggered an immunological response, as we observed a significant elevation of IL-6 in the NC group which were significantly reduced by Entelon150® treated group. Steroidal anti-inflammatory therapy significantly reduces the incidence of postoperative valve tissue rejection in patients, indicating that suppressing the valve-induced immunological response may improve the postoperative durability of bioprosthetic aortic valve implants [21]. Importantly, our data showed that Entelon150® treatment significantly lowered IL-6 levels, thus mitigating inflammation.

BMP-2 is a member of the transforming growth factor (TGF) superfamily and is known to be a master regulator of conventional and ectopic osteogenesis [22]. Alteration of BMP-2 reportedly aggravates skeletal and extraskeletal mineralization [23]. In addition, BMP-2 plays a critical role in vascular disease, including atherosclerosis and plaque instability through its effects on vascular inflammation. BMP-2 also regulates vascular oxidative stress and vascular calcification by stimulating osteogenesis in vascular smooth muscle cells [24]. Furthermore, IL-6 activity is strongly associated BMP-2 expression [3, 14] and calcification [25]. In this study we found that calcium content and IL-6 expression were significantly lowered in the bovine pericardium of the Entelon150®-treated group. To elucidate the underlying mechanism involved, we evaluated BMP-2 expression in implanted tissue. Interestingly, Entelon150® treatment significantly lowered BMP-2 expression, demonstrating its therapeutic molecular effects. OPN is an extracellular matrix glycoprotein mainly taking part in bone morphogenesis, bio-mineralization and calcification. OPN is produced as a cytokine in activated T cells and macrophages, demonstrating that OPN plays an important role modulating inflammation. During the healing process or under pro-inflammatory conditions, OPN expression is elevated near inflammatory cells. OPN is reportedly associated with inflammation, atherosclerosis, and vascular calcification [26]. We also found that along with IL-6 and calcification, OPN expression was increased in the implanted tissue and was non significantly lowered by Entelon150® administration. Additionally, α-SMA expression is used as a measure of tissue fibrosis. Calcification and fibrosis have many common features such as risk factors and have histopathological lesions with similar pathogenic pathways and mediators. The factors initiating calcification include inflammation, cell injury, and tissue infiltration by inflammatory cells, lipids, cytokines, and reactive oxygen species and the overexpression of α-SMA in calcified tissue has also been reported [27, 28]. Consistent with these findings, the expression α-SMA in the bovine pericardium was lowered by Entelon150® treatment; however, this difference was not statistically significant.

We have previously found that the angiotensin II type 1 receptor blocker losartan attenuates bioprosthetic valve leaflet calcification in a rabbit model of intravascular implantation [3]. Calcification of bovine pericardium appears unrelated to specific mechanisms, but rather appears related to reduced inflammation and substances like IL-6, BMP-2, and OPN. Any substance that lowers inflammation through IL-6, BMP-2, and OPN may help prevent calcification. From this point of view, Entelon150®, which consists of grape seed extract, will be more powerful than other synthetic medications at preventing calcification.

Limitations

The limitations of this study are its relatively low number and the uncertain mechanism of degenerative calcification in our beagle dog model of intravascular bovine pericardium implantation. It is unclear whether the mechanisms in our model are similar to the mechanisms underlying calcification of bovine pericardium in humans. However, we previously compared five implantation methods in a rabbit model and found that the intravenous implantation model most closely resembled bioprosthetic valve made of bovine pericardium calcification in humans. Furthermore, we reported that the calcium content was higher in intravenous implants than in arterial patch implants [29]. We performed our experiments in beagle dogs rather than rabbits; however, we think our results are consistent with the findings in our rabbit intravascular model.

Conclusion

We found that Entelon 150® significantly attenuated post-implant degenerative changes inflammation and calcium deposition in a beagle dog of intravascular bovine pericardium implantation model. Further observations are required to assess the effects of Entelon 150® on native vessel calcification in another animal model.

Supporting information

S1 Raw images

(PDF)

Click here for additional data file. (234.2KB, pdf)

Data Availability

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

Funding Statement

This study was supported by a Korea University Grant (K1924931).

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  • 29.Kim D, Park HK, Park YH, Lee SH, Joo HC, Lee S, et al. Degenerative calcification of pericardial bioprostheses: comparison of five implantation methods in a rabbit model. J Heart Valve Dis 2015. September; 24(5):621–8. [PubMed] [Google Scholar]

Decision Letter 0

Andreas Zirlik

17 Nov 2020

PONE-D-20-18358

Entelon® (vitis vinifera seed extract) reduces inflammation and calcification in a beagle dog model of intravascular bovine pericardium implantation

PLOS ONE

Dear Dr. Shin,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but 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.

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We look forward to receiving your revised manuscript.

Kind regards,

Andreas Zirlik, MD

Academic Editor

PLOS ONE

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2. At this time, we request that you  please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

(1) Please state the source of the dogs used in the study  

(2) Please include the method of euthanasia

(3) Please describe the post-operative care received by the dogs, including the frequency of monitoring and the criteria used to assess animal health and well-being.  

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We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

(1) You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

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In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

(2) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

4. Please provide the source, product number and any lot numbers of the Entelon150 purchased from Hanlim Pharm. Co., Ltd  for your study.”

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. 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: Partly

Reviewer #2: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. 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: No

Reviewer #2: Yes

**********

4. 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

**********

5. 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: Dear Editor!

Thank you for the opportunity to review this interesting paper! Choi and colleagues evaluated the local reaction regarding inflammation and calcification after bovine patch implantation and the potential modification with systemic treatment of Entelon over six weeks, which is a grape seed extract.

While the findings are interesting, I do have some concern regarding the applicability and interpretation of the results which needs to be addressed:

Major:

- The authors conclude that the systemic treatment reduced the calcification in the patch area. However, despite reduced calcium concentration, no direct calcification could be shown. This would also be very early after six weeks. Therefore, I would suggest to change the conclusion and drive the attention towards the changes is inflammatory reaction.

- In general, the concept of systemic treatment to hinder degeneration of an implanted bioprosthesis is interesting and needs further discussion. It seems obvious that for example full immunosuppression would avoid calcification of aortic or pulmonary native homograft implantation, as this is not seen after heart transplantation. However, nobody would treat a patient after homograft implantation with full immunosuppression due to the side effects of a life-long therapy. Therefore, the current approach is to avoid immunoreaction towards homografts by decellularization to avoid the immune reaction. If a systemic therapy needs to be administered after bovine valve implantation to hinder tissue calcification, this therapy requires to have almost no side effects over 10-20 years, which is the current typical life-time of a bioprosthetic valve. Therefore, the applied dose of Entelon and the associated expected risk profile in human administration needs to be addressed and discussed. Furthermore, alternative therapies like statins have previously been evaluated and needs to be addressed.

- The authors mention the low case number in the limitation session. This is indeed a critical issue. A very strong underlying effect is to be expected if significant differences were observed with 4 animals per group. A higher sample size would be beneficial.

- It seems interesting that Entelon is reducing BMP-2 concentration in the affected tissue as the potential underlying mechanism. Was this effect previously observed? What is the potential mechanism to change BMP-2 activity?

Minor:

- The patch was implanted in the venous position. However, an arterial patch would have more stress and a potential earlier degeneration. Why was a venous place chosen?

Reviewer #2: Dear Authors,

selecting the topic “Inflammation and calcification in the setting of bio-prosthetic valve and other substitute heart valve implantation failure, you are taking on an ongoing problem in current state-of-the-art medical therapy.

The authors are able to show that a substance known to reduce overall inflammatory signaling is able to reduce calcium content as well as the inflammation level in the specific setting of intravenous bovine pericardium implants in dogs.

The study itself is well-designed and overall, the manuscript is well written and presents the findings in a comprehensive manner. The figures present the clinical as well as in-vitro data in a concise way, while surely benefitting from some more details.

However, there are several points that need clarification which will surely benefit the manuscript and make it more easy to follow.

1. Line 260ff: The conclusion should be rephrased. In line 171, the authors mention there were no calcified lesions detectable in any of the animals. Also the follow up of only 6 weeks is rather short term whereas degenerative calcification is a long term process. On the other hand, the significant attenuation of inflammation is not mentioned

2. Line 112ff: When collecting the sample you describe storing the prosthetic — was this with or without the surrounding venous tissue? Please clarify where lesions and calcification were examined. Right now, it remains unclear if this analysis was performed on the same tissues.

3. Western Blotting:

Line 147: how was the protein content of samples quantified? Did you perform a Bradford Assay or some different method?

4. Line 153: I suspect you washed the membranes after staining with secondary antibody? Please correct or elaborate further on the procedure.

5. Line 167ff: Please specify the time point(s) at which body weight and vascular patency were examined in the results section. At the moment there is only 1 side note in the methods section under “sample collection”. Also, I can only assume the CT scan was performed at the same time as ultrasound examination took place? Please specify

6. Line 171: Please define “calcified lesion”. You state that there were no lesions in the implant in either group. However, overall Calcium content was higher in the negative control group. Are the measured Calcium levels after bovine patch implantation higher as compared to untreated veins?

7. Line 243: please specify which “certain mechanism” you are talking about. In its current form, this statement provides no value to the reader.

8. Line 246: It is unclear to me as to why a phytochemical substance should be more powerful than other systemic agents? You state that “any substance” lowering inflammation through a given signaling pathway may help prevent calcification. Please rephrase.

9. Figure 1:

Please label the anatomical landmarks mentioned in the manuscript and relevant for understanding figure 2 B+C. Also, it would be helpful to indicate the location of incision and subsequent bovine pericardium patch implantation into the schematic drawing to exclude any ambiguity.

10. Figure2:

Please label the vessels and anatomical structures shown (see comment for figure1). Also, although there are no occlusions visible, one has the impression that in the control-group, the vessels shown have an overall smaller and more irregular diameter. This should be discussed in the manuscript.

**********

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Reviewer #1: No

Reviewer #2: No

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PLoS One. 2021 Mar 4;16(3):e0235454. doi: 10.1371/journal.pone.0235454.r002

Author response to Decision Letter 0

14 Jan 2021

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and

https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

2. At this time, we request that you please report additional details in your Methods section regarding animal care, as per our editorial guidelines:

� Please state the source of the dogs used in the study

⇒ According to this comment, I deleted the latter “multiple The source of dogs is added, sorry for this mistake

“Eight healthy male beagle dogs (20 weeks old; mean body weight 9.76 ± 0.32 kg, range 8.00–10.80 kg) were used in this study which were purchased from ORIENT BIO Inc. (Seongnam, Gyeonggi-do, Republic of Korea)”.

� (2) Please include the method of euthanasia

⇒ According to this comment, Euthenesia methods are added. We are sorry for this mistake-

“Six weeks after surgery and immediately after measuring vascular patency, the animals were euthanized by propofol (10 mg/kg, IV) and pentobarbital (50 mg/kg, IV)”.

� (3) Please describe the post-operative care received by the dogs, including the frequency of monitoring and the criteria used to assess animal health and well-being.

⇒ Dear respected editor, According to your comment postoperative care and monitoring are included details

“Postoperatively, intramuscular enrofloxacine (10 mg/kg, bid), intravenous cephradine (30 mg/kg bid or tid), intravenous tramadol (2–3 mg/kg bid or tid) and intravenous cimetidine (10 mg/kg, bid) were performed for three days [12]. Respiratory rate was monitored by the movement of the abdominal and chest wall (breath/min) twice daily. Heart rate and ECG was recorded with a Cardiofax ECG-9020 electrocardiograph (Nihon Kohden, Tokyo, Japan) once daily for three days [13]. Beside these normal clinical sings were closely monitored daily until end of the experiment to identify any abnormality due to surgery or any side effect for administering Entelon150 including feeding and drinking behavior, urination and defecation frequency, bleeding, salivating, vomiting, abnormality or redness of skin and eye color, abnormal sound and abnormal movement but none were observed in entire experimental period.”.

3. We note that Figure 1 in your submission contains copyrighted images. All PLOS content is published under the Creative Commons Attribution License (CC BY 4.0), which means that the manuscript, images, and Supporting Information files will be freely available online, and any third party is permitted to access, download, copy, distribute, and use these materials in any way, even commercially, with proper attribution. For more information, see our copyright guidelines: http://journals.plos.org/plosone/s/licenses-and-copyright.

We require you to either (1) present written permission from the copyright holder to publish these figures specifically under the CC BY 4.0 license, or (2) remove the figures from your submission:

(1) You may seek permission from the original copyright holder of Figure 1 to publish the content specifically under the CC BY 4.0 license.

We recommend that you contact the original copyright holder with the Content Permission Form (http://journals.plos.org/plosone/s/file?id=7c09/content-permission-form.pdf) and the following text:

“I request permission for the open-access journal PLOS ONE to publish XXX under the Creative Commons Attribution License (CCAL) CC BY 4.0 (http://creativecommons.org/licenses/by/4.0/). Please be aware that this license allows unrestricted use and distribution, even commercially, by third parties. Please reply and provide explicit written permission to publish XXX under a CC BY license and complete the attached form.”

Please upload the completed Content Permission Form or other proof of granted permissions as an "Other" file with your submission.

In the figure caption of the copyrighted figure, please include the following text: “Reprinted from [ref] under a CC BY license, with permission from [name of publisher], original copyright [original copyright year].”

(2) If you are unable to obtain permission from the original copyright holder to publish these figures under the CC BY 4.0 license or if the copyright holder’s requirements are incompatible with the CC BY 4.0 license, please either i) remove the figure or ii) supply a replacement figure that complies with the CC BY 4.0 license. Please check copyright information on all replacement figures and update the figure caption with source information. If applicable, please specify in the figure caption text when a figure is similar but not identical to the original image and is therefore for illustrative purposes only.

⇒ Dear respected editor, Fig. 1 is our original drawing. When we submited this manuscript in PLOS ONE first, there was asking to reveal our data previously in bioRxiv. Thus, we permitted it. According to bioRxiv preprint doi: https://doi.org/10.1101/2020.06.17.156695; this version posted June 17, 2020. The copyright holder for this preprint is the author/funder. who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. And the corresponding author is Hong Ju Shin. Fig. 1 is important picture of this manuscript. Dr. Shin made this picture with illustrator at first.

� 4. Please provide the source, product number and any lot numbers of the Entelon150 purchased from Hanlim Pharm. Co., Ltd for your study.”

⇒ The source and lot number is provided in material and methods section

“(Entelon150®; Lot number: RNR601, Hanlim Pharm. Co., Ltd. Yongin-si, Korea)”.

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

2. 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: Partly

Reviewer #2: Yes

________________________________________

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

Reviewer #1: Yes

Reviewer #2: Yes

________________________________________

3. 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: No

Reviewer #2: Yes

________________________________________

4. 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

________________________________________

5. 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:

Dear Editor! Thank you for the opportunity to review this interesting paper! Choi and colleagues evaluated the local reaction regarding inflammation and calcification after bovine patch implantation and the potential modification with systemic treatment of Entelon over six weeks, which is a grape seed extract. While the findings are interesting, I do have some concern regarding the applicability and interpretation of the results which needs to be addressed:

� Major:

The authors conclude that the systemic treatment reduced the calcification in the patch area. However, despite reduced calcium concentration, no direct calcification could be shown. This would also be very early after six weeks. Therefore, I would suggest to change the conclusion and drive the attention towards the changes is inflammatory reaction.

- In general, the concept of systemic treatment to hinder degeneration of an implanted bioprosthesis is interesting and needs further discussion. It seems obvious that for example full immunosuppression would avoid calcification of aortic or pulmonary native homograft implantation, as this is not seen after heart transplantation. However, nobody would treat a patient after homograft implantation with full immunosuppression due to the side effects of a life-long therapy. Therefore, the current approach is to avoid immunoreaction towards homografts by decellularization to avoid the immune reaction. If a systemic therapy needs to be administered after bovine valve implantation to hinder tissue calcification, this therapy requires to have almost no side effects over 10-20 years, which is the current typical life-time of a bioprosthetic valve. Therefore, the applied dose of Entelon and the associated expected risk profile in human administration needs to be addressed and discussed. Furthermore, alternative therapies like statins have previously been evaluated and needs to be addressed.

⇒ After consideration your comments we have changes “Title”, and rewrite “Discussion” and “Conclusion”.

Title: “Entelon® (vitis vinifera seed extract) reduces degenerative changes in bovine pericardium valve leaflet in a dog intravascular implant model”

Discussion:

“Calcification or degenerative changes of implant valve in human usually takes longtime [16]. However, degeneration was observed comparatively with in shorter time in experimental implant model; evaluated after 14 and 30 days [17], 4 and 12 weeks [14], 3 weeks [18], 6 weeks [3,19, 29] implantation. Subcutaneous and intramuscular implantation models are usually performed to investigate BHV degenerative changes or calcification (14,17,18,19). Unfortunately, subcutaneous and intramuscular models are not appropriate as graft tissue experienced hemodynamic stress due to the direct blood contact or circulating factors inside the heart or great vessels. Therefore, intravascular implant model became popular model [3,29]. We therefore performed intravascular implant model”.

Conclusion:

“We found that Entelon 150® significantly attenuated post-implant degenerative changes inflammation and calcification in a beagle dog of intravascular bovine pericardium implantation model. Further observations are required to assess the effects of Entelon 150® on native vessel calcification in another animal model”.

� - The authors mention the low case number in the limitation session. This is indeed a critical issue. A very strong underlying effect is to be expected if significant differences were observed with 4 animals per group. A higher sample size would be beneficial.

⇒ Dear respected reviewer we also mentioned it in our limitation part

� - It seems interesting that Entelon is reducing BMP-2 concentration in the affected tissue as the potential underlying mechanism. Was this effect previously observed? What is the potential mechanism to change BMP-2 activity?

⇒ Dear respected reviewer we have mentioned the underlying mechanism and relationship of BMP-2 with inflammation and calcification in “Discussion”-

“BMP-2 is a member of the transforming growth factor (TGF) superfamily and is known to be a master regulator of conventional and ectopic osteogenesis [17]. Alteration of BMP-2 reportedly aggravates skeletal and extraskeletal mineralization [18]. In addition, BMP-2 plays a critical role in vascular disease, including atherosclerosis and plaque instability through its effects on vascular inflammation. BMP-2 also regulates vascular oxidative stress and vascular calcification by stimulating osteogenesis in vascular smooth muscle cells [19]. Furthermore, IL-6 activity is strongly associated BMP-2 expression [3,12] and calcification [20]. In this study we found that calcium content and IL-6 expression were significantly lowered in the bovine pericardium of the Entelon150®-treated group. To elucidate the underlying mechanism involved, we evaluated BMP-2 expression in implanted tissue. Interestingly, Entelon150® treatment significantly lowered BMP-2 expression, demonstrating its therapeutic molecular effects.

� Minor:

The patch was implanted in the venous position. However, an arterial patch would have more stress and a potential earlier degeneration. Why was a venous place chosen?

⇒ Dear respected reviewer, we previously compared five implantation methods in a rabbit model and found that the calcium content was higher in intravenous implants than in arterial patch implants (J Heart Valve Dis 2015;24:621-8). Actually it is technically difficult to implant intra-arterial patch in small carotid artery (2~3mm size). Therefore, we chose intravenous implants model.

Reviewer #2:

Dear Authors, selecting the topic “Inflammation and calcification in the setting of bio-prosthetic valve and other substitute heart valve implantation failure, you are taking on an ongoing problem in current state-of-the-art medical therapy. The authors are able to show that a substance known to reduce overall inflammatory signaling is able to reduce calcium content as well as the inflammation level in the specific setting of intravenous bovine pericardium implants in dogs.

The study itself is well-designed and overall, the manuscript is well written and presents the findings in a comprehensive manner. The figures present the clinical as well as in-vitro data in a concise way, while surely benefitting from some more details. However, there are several points that need clarification which will surely benefit the manuscript and make it more easy to follow.

� 1. Line 260ff: The conclusion should be rephrased.

⇒ We have rephrased conclusion. Could you please see below -

Conclusion:

“We found that Entelon 150® significantly attenuated post-implant degenerative changes inflammation and calcium deposition in a beagle dog of intravascular bovine pericardium implantation model. Further observations are required to assess the effects of Entelon 150® on native vessel calcification in another animal model”.

In line 171, the authors mention there were no calcified lesions detectable in any of the animals.

⇒ Dear respected reviewer it was our mistake; we would like to say any type of occlusion was not observed. We have corrected the sentence. Could you please see below -

“Although there are no occlusions visible around the vessel or in the implants in either control or Entelon150®-treated groups, the vessels shown have an overall smaller and more irregular diameter might be for suturing effect (Fig.2)”.

Also the follow up of only 6 weeks is rather short term whereas degenerative calcification is a long term process.

⇒ Thank you for your comments. As you commented, We changed “Title”, and rewrite

“Discussion” and “Conclusion”.

Title: “Entelon® (vitis vinifera seed extract) reduces degenerative changes in bovine pericardium valve leaflet in a dog intravascular implant model”

Discussion:

“Calcification or degenerative changes of implant valve in human usually takes longtime [16]. However, degeneration was observed comparatively with in shorter time in experimental implant model; evaluated after 14 and 30 days [17], 4 and 12 weeks [14], 3 weeks [18], 6 weeks [3,19, 29] implantation. Subcutaneous and intramuscular implantation models are usually performed to investigate BHV degenerative changes or calcification (14,17,18,19). Unfortunately, subcutaneous and intramuscular models are not appropriate as graft tissue experienced hemodynamic stress due to the direct blood contact or circulating factors inside the heart or great vessels. Therefore, intravascular implant model became popular model [3,29]. We therefore performed intravascular implant model”.

Conclusion:

“We found that Entelon 150® significantly attenuated post-implant degenerative changes inflammation and calcification in a beagle dog of intravascular bovine pericardium implantation model. Further observations are required to assess the effects of Entelon 150® on native vessel calcification in another animal model”.

On the other hand, the significant attenuation of inflammation is not mentioned

⇒ Dear respected reviewer we have now corrected and mentioned in several times that treatment reduced inflammation. Could you please see below -

Result:

“Western blot analysis also revealed that IL-6 levels in the Entelon150®-treated group (55.36±5.49%) were significantly lower (P < 0.001) than NC group (100.00±10.30%) indicating the significant attenuation of inflammation”.

“Histopathological examination revealed infiltration of chronic inflammatory cells such as fibroblasts and macrophages around the graft in all groups. However, the inflammation level of the Entelon150®-treated group (1.50±0.58%) was significantly lower (P < 0.001) than the NC group (2.25±0.96%). In particular, a basophilic substance presumed to be the earliest sign of calcium deposition was observed in the NC group between the intercellular matrixes of the peri-graft tissue (Fig 6)”.

Discussion:

“In this study we demonstrated that Entelon150® treatment had two effects: significantly attenuating both inflammation and calcification in a beagle dog model of intravascular bovine pericardial implantation”.

“Indeed, immunological rejection of heterologous tissue is a challenging medical problem contributing to post-implant xenograft degeneration [1914]. Our histopathological results are consistent with previous studies observing infiltration of chronic inflammatory cells such as fibroblasts and macrophages around the graft. The inflammation levels of the Entelon150®-treated group was significantly lower than the negative control group. In addition, a basophilic substance, presumed to be the earliest sign of calcium deposition, was observed between the intercellular matrixes of the peri-graft tissue in the negative control group. The basophilic staining using the H & E stain method indicated calcium deposition [2015]. The results suggest that administration of Entelon150® lowered inflammation levels and inhibited calcium deposition in the tissues surrounding the graft”.

“In addition, we found that bovine pericardium triggered an immunological response, as we observed a significant elevation of IL-6 in the NC group which were significantly reduced by Entelon150® treated group. Steroidal anti-inflammatory therapy significantly reduces the incidence of postoperative valve tissue rejection in patients, indicating that suppressing the valve-induced immunological response may improve the postoperative durability of bioprosthetic aortic valve implants [2116]. Importantly, our data showed that Entelon150® treatment significantly lowered IL-6 levels, thus mitigating inflammation.”

� 2. Line 112ff: When collecting the sample, you describe storing the prosthetic — was this with or without the surrounding venous tissue? Please clarify where lesions and calcification were examined. Right now, it remains unclear if this analysis was performed on the same tissues.

⇒ Dear respected reviewer we rewrote the according to your nice comment-

“Only implanted prosthetic was collected from the right external jugular vein and connective tissues around the implanted prosthetic was carefully removed and then stored in a cryogenic freezer maintained at about -80 ° C until calcium quantification. However, half of the left external jugular vein along with connective tissue was fixed in a 10% neutral buffered formalin solution for histopathological examination, and from the other half part only prosthetic was stored in a cryogenic freezer maintained at -80 ° C for western blotting”.

� - 3. Western Blotting: Line 147: how was the protein content of samples quantified? Did you perform a Bradford Assay or some different method?

⇒ We have added the assay method. Sorry for this mistake

“Cryogenically frozen samples were homogenized using RIPA buffer, after which proteins were extracted and quantified with a protein assay kit (Bio-Rad, CA, USA)”.

� 4. Line 153: I suspect you washed the membranes after staining with secondary antibody? Please correct or elaborate further on the procedure.

⇒ Dear respected reviewer we have already mentioned it. Could you please see below-

“After the primary antibody reaction, the membrane was exposed to the secondary antibody (diluted 1: 10,000). After the reaction was complete, the cells were washed with PBS-T buffer (0.5% Tween 20 in phosphate buffered saline) and the sample was developed using an enhanced chemiluminescence (ECL) reagent for immunoblot analysis”.

� - 5. Line 167ff: Please specify the time point(s) at which body weight and vascular patency were examined in the results section. At the moment there is only 1 side note in the methods section under “sample collection”.

Also, I can only assume the CT scan was performed at the same time as ultrasound examination took place? Please specify

⇒ Dear respected reviewer by following your nice comment we have mentioned the time schedule of body weight measurement, CT-scan, angiography evaluation. Could you please see below –

Time schedule of Body weight measurement

“Body weight was measured just before starting experiment and then once a week until six weeks (at o, 1, 2, 3, 4, 5 and 6 weeks) (Fig 2)”.

Time schedule of CT-scan and angiography evaluation

“Vascular patency was checked using the color doppler mode of an ultrasonic device (LOGIQ e Ultrasound; GE Healthcare, Fairfield, USA) at 4 weeks and 6 weeks after the bioprosthetic implantation. Angiography was also performed with a bolus intravenous injection of 2 ml/kg iohexol (Omnipaque™, 300 mg I/ml; GE Healthcare) using a CT-scanner (CT; 16-channel multidetector; Bright Speed Elite, GE Healthcare, Fairfield, CT, USA) for additional confirmation just after ultrasonograpy and immediately before sacrifice at 6 weeks”.

� 6. Line 171: Please define “calcified lesion”. You state that there were no lesions in the implant in either group. However, overall Calcium content was higher in the negative control group. Are the measured Calcium levels after bovine patch implantation higher as compared to untreated veins?

⇒ Dear respected reviewer it was our mistake; we would like to say any type of occlusion was not observed. We have corrected the sentence. Could you please see below –

“Although there are no occlusions visible around the vessel or in the implants in either control or Entelon150®-treated groups, the vessels shown have an overall smaller and more irregular diameter might be for suturing effect (Fig.2)”.

� 7. Line 243: please specify which “certain mechanism” you are talking about. In its current form, this statement provides no value to the reader.

⇒ Dear respected reviewer, I meant that calcification is not related to specific mechanism, but related to lower inflammation including IL-6,BMP-2, Ostenopontin.

Therefore, I switched certain to specific.

We have discussed with the references the underlying mechanism and relationship of IL6, BMP-2 and OPN with inflammation and calcification in “Discussion”-

“IL-6:

In addition, we found that bovine pericardium triggered an immunological response, as we observed a significant elevation of IL-6 in the NC group. Steroidal anti-inflammatory therapy significantly reduces the incidence of postoperative valve tissue rejection in patients, indicating that suppressing the valve-induced immunological response may improve the postoperative durability of bioprosthetic aortic valve implants [21]. Importantly, our data showed that Entelon150® treatment significantly lowered IL-6 levels, thus mitigating inflammation.

BMP-2:

BMP-2 is a member of the transforming growth factor (TGF) superfamily and is known to be a master regulator of conventional and ectopic osteogenesis [22]. Alteration of BMP-2 reportedly aggravates skeletal and extraskeletal mineralization [23]. In addition, BMP-2 plays a critical role in vascular disease, including atherosclerosis and plaque instability through its effects on vascular inflammation. BMP-2 also regulates vascular oxidative stress and vascular calcification by stimulating osteogenesis in vascular smooth muscle cells [24]. Furthermore, IL-6 activity is strongly associated BMP-2 expression [3,14] and calcification [25]. In this study we found that calcium content and IL-6 expression were significantly lowered in the bovine pericardium of the Entelon150®-treated group. To elucidate the underlying mechanism involved, we evaluated BMP-2 expression in implanted tissue. Interestingly, Entelon150® treatment significantly lowered BMP-2 expression, demonstrating its therapeutic molecular effects. OPN:

OPN is an extracellular matrix glycoprotein mainly taking part in bone morphogenesis, bio-mineralization and calcification. OPN is produced as a cytokine in activated T cells and macrophages, demonstrating that OPN plays an important role modulating inflammation. During the healing process or under pro-inflammatory conditions, OPN expression is elevated near inflammatory cells. OPN is reportedly associated with inflammation, atherosclerosis, and vascular calcification [26]. We also found that along with IL-6 and calcification, OPN expression was increased in the implanted tissue and was non significantly lowered by Entelon150® administration.

α-SMA:

Additionally, α-SMA expression is used as a measure of tissue fibrosis. Calcification and fibrosis have many common features such as risk factors and have histopathological lesions with similar pathogenic pathways and mediators. The factors initiating calcification include inflammation, cell injury, and tissue infiltration by inflammatory cells, lipids, cytokines, and reactive oxygen species and the overexpression of α-SMA in calcified tissue has also been reported [27,28]. Consistent with these findings, the expression α-SMA in the bovine pericardium was lowered by Entelon150® treatment; however, this difference was not statistically significant”.

� 8. Line 246: It is unclear to me as to why a phytochemical substance should be more powerful than other systemic agents? You state that “any substance” lowering inflammation through a given signaling pathway may help prevent calcification. Please rephrase.

⇒ Dear respected reviewer we mentioned it in the “Introduction part”. Could you please see below –

“Several approaches to reduce calcification have been attempted, including systemic anti-calcification agent administration. However, many of these approaches have been either ineffective or have produced unwanted side effects [3]. To overcome this drawback, studies on the complementary treatment of calcification have focused on traditional herbal medicines recently [4-6]”.

� 9. Figure 1: Please label the anatomical landmarks mentioned in the manuscript and relevant for understanding figure 2 B+C. Also, it would be helpful to indicate the location of incision and subsequent bovine pericardium patch implantation into the schematic drawing to exclude any ambiguity.

⇒ Dear respected reviewer we have corrected the figure according to your suggestion .

Fig1. Anatomical diagram of a beagle’s jugular vein during the implantation procedure.

Yellow star indicates the incision site of jugular vein

� 10. Figure2: Please label the vessels and anatomical structures shown (see comment for figure1). Also, although there are no occlusions visible, one has the impression that in the control-group, the vessels shown have an overall smaller and more irregular diameter. This should be discussed in the manuscript.

⇒ Dear respected reviewer we corrected figure-2 and we mentioned it in the “Result sectiont”. Could you please see below –

“Although there are no occlusions visible around the vessel or in the implants in either control or Entelon150®-treated groups, the vessels shown have an overall smaller and more irregular diameter might be suturing effect (Fig.2)”.

Fig 2. Effects of bioprosthetic valve implantation and Entelon® treatment on body weight and vascular patency as measured by CT-Scan.

Arrow indicates the incision and implanted site in jugular vein.

________________________________________

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Decision Letter 1

Andreas Zirlik

11 Feb 2021

Entelon® (vitis vinifera seed extract) reduces degenerative changes in bovine pericardium valve leaflet in a dog intravascular implant model

PONE-D-20-18358R1

Dear Dr. Shin,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Reviewer #2: All comments have been addressed

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Reviewer #2: Yes

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

Reviewer #2: I Don't Know

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Reviewer #2: Yes

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Reviewer #2: Dear Authors,

thank you again for this study on Inflammation and calcification in the setting of bio-prosthetic valve and other substitute heart valve implantation failure, which is an ongoing problem in current state-of-the-art medical therapy

All of my points of concern raised in the initial submit were dealt with thoroughly.

Revising your manuscript has clearly added to the readability, putting your significant findings into focus and better clinical context.

Also, the revised figures now help in visualizing the implantation site and make the figures more consistent.

This certainly made your manuscript even better suited for practicing cardio-thoracic surgeons and cardiologists.

**********

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If you choose “no”, your identity will remain anonymous but your review may still be made public.

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Reviewer #2: No

Acceptance letter

Andreas Zirlik

23 Feb 2021

PONE-D-20-18358R1

Entelon (vitis vinifera seed extract) reduces degenerative changes in bovine pericardium valve leaflet in a dog intravascular implant model

Dear Dr. Shin:

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.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Univ. Prof. Dr. Andreas Zirlik

Academic Editor

PLOS ONE

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