Published online Sep 12, 2023.
https://doi.org/10.4070/kcj.2023.0120
Entelon150® (Vitis vinifera Seed Extract) Attenuates Degenerative Changes in Intravascular Valve Prostheses in Rabbits
, MD, PhD,1
JungHyeok Seo, PhD,2
Sokho Kim, PhD,3
Md. Mahbubur Rahman, PhD,4
and Hong Ju Shin, MD, PhD5
Author's summary
Entelon150® (Vitis vinifera seed extract), losartan, and rosuvastatin have been shown to be effective in reducing calcification and inflammation of bovine pericardium implants. However, no study has compared the effects of the drugs on bioprosthetic heart valve (BHV). In this comparative study using a rabbit model, Entelon150® exhibited the significant effect, ultimately reducing calcification and inflammation in the intravascular bovine pericardium. Entelon150® could be a promising therapeutic alternative for reducing BHV inflammation and calcification.
Abstract
Background and Objectives
The therapeutic strategy for inflammation and degenerative calcification is of utmost importance for bioprosthetic heart valve (BHV) implanted patients. The purpose of this study was to compare the anti-inflammatory and anti-calcification effects of Entelon150® (grape seed extract), losartan, and rosuvastatin, in a rabbit model of intravascular BHV leaflet implantation in bovine pericardium.
Methods
A total of 28 rabbits were implanted with BHV leaflet in the external jugular veins. The Entelon150® group was administered 7.7 mg/kg Entelon150® twice daily for 6 weeks after surgery. The losartan and rosuvastatin groups received 5.14 mg/kg and 1 mg/kg, respectively, once per day. The control group received 1 ml of saline once daily. And then, calcium concentration was measured in the implanted BHV, and histological and molecular analyses were performed on the surrounding tissues.
Results
The calcium content of the implanted tissue in the Entelon150® group (0.013±0.004 mg/g) was lower than that in the control group (0.066±0.039 mg/g) (p=0.008). The losartan (0.024±0.016 mg/g, p=0.032) and rosuvastatin (0.022±0.011 mg/g, p=0.032) groups had lower calcium content than the control group, and higher tendency than the Entelon150® group. Immunohistochemistry revealed that the expressions of bone morphogenic protein 2 (BMP2), S-100, and angiotensin II type 1 receptor in the Entelon150® group showed lower tendency than those in the control group. The protein expression levels of BMP2 were reduced in the Entelon150® group compared with those in the control group.
Conclusions
Entelon150® exhibited a significant effect, similar to other drugs, in reducing calcification and inflammation in the intravascular bovine pericardium.
Graphical Abstract
INTRODUCTION
Bioprosthetic heart valve (BHV) has been used since the 1960s.1) Approximately ten years later, BHV was developed and remains widely used today.1) BHV is associated with a low risk of thromboembolism, but can cause tissue degeneration. BHV is also known to have relatively weak durability compared to mechanical valves.1), 2) This degenerative change of BHV is caused by the complex action of several factors, such as calcification, immunologic response, and mechanical stress.3), 4) Calcification is the most important cause of BHV failure.1)
Many attempts have been made to reduce calcification, including the development of anti-calcification medications; however, the results are not satisfactory and concerns exist regarding side effects. Based on previous animal experiments, losartan (angiotensin II type 1 receptor [AIIR] blocker) and rosuvastatin (HMG-CoA reductase inhibitors) exert a reducing effect on BHV calcification.5), 6), 7) These drugs can also be used to prolong the lifespan of BHV; however, the side effects associated with the long-term use of these drugs remain a concern.
The herbal product, Entelon150® (Vitis vinifera seed extract), was previously found to be effective at reducing post-implantation inflammation and calcification in the bovine pericardium transplanted into dogs, confirming its potential to extend the lifespan of the bovine pericardium.8) However, a comparative study with other prescribed drugs in BHV-implanted patients is necessary to validate the efficacy of Entelon150®. An evaluation of the anti-inflammatory and anti-calcification effects of Entelon150® in BHV-implanted models in different animal species is also important to confirm its efficacy.
Therefore, the purpose of this study was to compare the anti-inflammatory and anti-calcification effects of losartan, rosuvastatin, and Entelon150® on BHV in rabbits.
METHODS
Ethical statement
All animal experiments were approved by the Institutional Animal Care and Use Committee of KNOTUS Co., Ltd., Incheon, Korea (Certificate No: IACUC 22-KE-0154, 03/17/2022).
Animals and experimental design
In this study, 28 New Zealand white rabbits (weight, 3.5±0.17 kg) were used. These rabbits were purchased from Hanlim laboratory animal Inc. (Hwaseong, Korea). The animals were housed separately in stainless-steel cages (500×L 800×H 500 mm) and raised in a controlled breeding environment (temperature, 23±3°C; relative humidity, 55±15%; ventilation frequency, 10–20 times/h; light cycle, 8 am to 8 pm; and illumination, 150 to 300 Lux). Animals were randomly divided into 4 groups of 7 animals each. The control group comprised a group not treated with test drugs after implantation.
Drug preparation and administration
The dosage of each drug was determined based on the animal equivalent dose.9) In the case of a 60 kg human, the daily doses for losartan, rosuvastatin, and Entelon150® were determined as 100 mg, 20 mg, and 300 mg, respectively. The Km factor for humans and rabbits was considered as 37 and 12, respectively. Accordingly, the dosage of the administered medication was calculated using the following formula:
The Entelon150® group was treated with 7.7 mg/kg Entelon150® (Hanlim Pharm., Yongin, Korea) twice daily for 6 weeks after implantation. Losartan potassium (MSD, NY, USA) and rosuvastatin calcium (MSN Laboratories Private Limited, Kondapur, India) were administered 5.14 mg/kg and 1 mg/kg orally once per day for 6 weeks after implantation. All animals were closely monitored during the experimental period. Animals showed symptoms of swelling in the facial area for approximately one week after the implantation; however, additional specific symptoms were not identified thereafter. Six weeks after implantation, samples of implanted BHV and their surrounding tissues were collected. And the animals were euthanized intravenously with 180 mg/kg sodium pentobarbital (Hanlim, Yongin, Korea).
Surgical procedure for implantation
Each animal was anesthetized intravenously with 5 mg/kg xylazine (Bayer AG, Leverkusen, Germany) and 10 mg/kg Zoletil® (VIRBAC, Carros, France). All animals received crystalloid solution (3 mL/kg/h) throughout the surgical procedure. After laying the animal in a dorsal recumbent position, the skin on the external jugular vein was incised and dissection was performed to expose the left external jugular vein from the sternohyoid muscle. After the administration of heparin (50 IU/kg, IV), the left jugular vein was temporarily blocked with bulldog clamps, and the left external jugular vein was cut longitudinally to a length of approximately 8 mm using a surgical blade (No. 11). Commercially available bovine pericardium BHV (Carpentier-Edwards Perimount Magna pericardial prosthesis; Edwards Lifesciences, Irvine, CA, USA), cut into a rectangular shape (2×4 mm), was fixed to the inner wall of the vein using a 7-0 polypropylene suture. The vascular incision site was then sutured using a 7-0 polypropylene suture (Figure 1). Bovine pericardium BHV was also implanted into the right external jugular vein using the same protocol described above. The schematic diagram shows the anatomy of the jugular vein in New Zealand white rabbits relevant to this surgical procedure (Figure 2). Heparin (50 IU/kg, IV) was administered 3 days after surgery.
Figure 1
Surgical images of intravascular BHV implantation. (A) Bovine pericardium BHV fixed to the inner wall of the vein. (B) Vascular incision site being sutured and confirmed blood flow.
BHV = bioprosthetic heart valve.
Figure 2
Schematic diagram of the anatomy of New Zealand white rabbit jugular vein.
Sample collection
Six weeks after implantation, animals were killed, and both the left and right external jugular veins on each side were harvested. Among the 7 animals in each group, 5 animals had the implanted BHV extracted from the right external jugular vein, with careful removal of surrounding connective tissues. The extracted samples were then stored in a cryogenic freezer at approximately −80°C for calcium measurement. Furthermore, in each group, the left external jugular vein of 5 animals was utilized for Western blot analysis, including the tissue surrounding the implanted BHV. The remaining 2 animals in each group had both external jugular veins, along with the connective tissue, fixed in a 10% neutral buffered formalin solution for subsequent hematoxylin and eosin (H&E) staining and immunohistochemical (IHC) analysis.
Measurement of calcium quantity
Quantitative calcium measurements were performed using inductively coupled plasma mass spectrometry (ICP-MS; Agilent, Santa Clara, CA, USA). After placing the sample in a sealed perfluoroalkoxy container and measuring the weight, 2 mL of ultrapure water and 2 mL of 70% nitric acid were added and then heated at 200°C with a graphite digestion system for 2 hours. After the container was cooled, ultrapure water was added and diluted to 10 g, and the solution was filtered using a 0.45 μm PTFE syringe filter. The diluted sample was used to measure the quantity of calcium.
Histopathological examination
Samples fixed with 10% NBF were pretreated with a tissue processor (ASP300S; Leica, Wetzlar, Germany) and embedded in paraffin (Tissue Tek TEC5; Sakura, Tokyo, Japan). Consecutive sections with a thickness of 4 μm, generated using a thinning machine (MULTICUTR; Leica), were attached to the coating slides (5116-20F; Muto, Tokyo, Japan). The samples were stained with H&E using an automatic staining machine (ST5010/CV5030; Leica). The infiltration of inflammatory cells into the tissue surrounding the implanted BHV was scored as follows: 0, none; 1, weak inflammatory cell infiltration; 2, moderate inflammatory cell infiltration; and 3, severe inflammatory cell infiltration.
IHC staining was performed using antibodies against BMP2, S-100, AIIR, and alpha smooth muscle actin (α-SMA). The primary antibodies were purchased from ABCAM (Waltham, MA, USA). The samples were incubated with BMP2 (BS-1012R, 1:8,000, 10 minutes), S-100 (ab22506, 1:800, 10 minutes), AIIR (ADI-905-743, 1:16,000, 1 hour), and α-SMA (ab7817, 1:1,000, 1 hour) and washed sufficiently with TBS-T. Thereafter, they were incubated with the secondary antibodies at room temperature for 30 minutes. IHC staining was performed via antigen exposure in a 65°C water bath for 16 hours using a 10-fold dilution of Tris-EDTA HIER solution buffer, followed by 0.3% H2O2 (10 minutes) and BSA blocking (30 minutes) after cooling at room temperature for 30 min. After sufficient washing, staining with DAB (K3468; Dako, Glostrup, Denmark) was performed for 30 seconds to 2 minutes via confirmation of color development under a microscope. Mayer's hematoxylin (S3309; Dako), a control dye, was dispensed and incubation was performed for 3 minutes. The stained slides were then washed, dehydrated, cleared, and mounted. Subsequently, 3 images around the implanted BHV were captured at magnifications of 40, 200, and 400 using a microscope (Axio Scope.A1; Carl Zeiss, Oberkochen, Germany). For the IHC image analysis, the positive staining area was compared to the total area at the region of interest, which was around the junction of the BHV and the surrounding tissue. This comparison was performed using an analysis program (Zen 3.4 image program; Carl Zeiss) at a scale of 400×, where the scale bar indicated 50 µm.
Western blot analysis
Ultra-low-temperature frozen samples (vascular tissue surrounding the implanted BHV) were homogenized using RIPA buffer. Thereafter, the protein was extracted and quantified. A sample for electrophoresis was prepared based on the quantified value, and electrophoresis was performed on a 10–14% acrylamide gel for 120 minutes. After the protein was transferred onto a polyvinylidene difluoride (PVDF) membrane, non-specific protein was removed using a blocking buffer. BMP2, interleukin-6 (IL-6), osteopontin (OPN), and β-actin primary antibodies were purchased from ABCAM, diluted to 1:1,000, and dispensed onto the PVDF membrane; the membrane was then incubated with these antibodies at approximately 4°C for more than 6 hours. After the first antibody reaction, the membrane was incubated with the secondary antibody diluted to 1:10,000. After the reaction was completed, the membrane was washed with PBS-T buffer (0.5% Tween 20 in phosphate-buffered saline). The sample was detected using an enhanced chemiluminescence (ECL) reagent, and analyzed using an image analysis device (iBright; Invitrogen, Waltham, MA, USA). β-actin was used as an intrinsic control. The protein expression based on the ECL signal was quantified using pixel density analysis.
Statistical analysis
The results of this study were analyzed using parametric multiple parallel procedures or non-parametric multiple parallel procedures. For parametric multiple comparisons, the data were assumed to be normal and were assessed using 1-way analysis of variance. If the results were significant, a post-test was performed using Dunnett's multiple comparison test to determine the significant differences between groups. For nonparametric multiple comparisons, the Kruskal-Wallis H-test was conducted, and if the results were significant, a post-test was performed using the Mann-Whitney U test to determine the significant differences between groups. Statistical analysis was performed using Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA), and p values less than 0.05 were considered statistically significant.
RESULTS
Vascular patency
To evaluate the patency of the external jugular vein after BHV implantation, we performed vascular contrast-enhanced computed tomography (Alexion; Toshiba, Tokyo, Japan) using iohexol (2 mL/kg, IV) at 4 weeks post BHV implantation. The results showed that out of 14 external jugular veins in each group, the control group had 3 veins occluded, the Entelon150® group had 2 veins occluded, the losartan group had 4 veins occluded, and the rosuvastatin group had 3 veins occluded.
Entelon150®, losartan, and rosuvastatin significantly reduce calcium content in implanted bioprosthetic heart valve
The Ca2+ quantity in the implanted BHV from the Entelon150®-, losartan-, and rosuvastatin-treated groups was significantly decreased compared to that in BHV from the non-treated control group (Figure 3). The calcium content of the implanted tissue in the Entelon150® group (0.013±0.004 mg/g) was significantly lower than that in the control group (0.066±0.039 mg/g) (p=0.008). The losartan group (0.024±0.016 mg/g, p=0.032) and rosuvastatin (0.022±0.011 mg/g, p=0.032) group had a lower calcium content than the control group, and a higher calcium content tendency than the Entelon150® group. However, no significant differences were found among the Entelon150®, losartan, and rosuvastatin groups.
Figure 3
Calcium concentration in the implanted BHV tissue in each group. Calcium levels were measured 6 weeks after implantation. The calcium levels in the control group were significantly higher than those in the Entelon, losartan, and rosuvastatin groups. Data are presented as mean±standard deviation.
BHV = bioprosthetic heart valve.
Entelon150®, losartan, and rosuvastatin reduce the infiltration of inflammatory cells around the implanted bioprosthetic heart valve vascular tissue
Infiltration of inflammatory cells, such as fibroblasts and macrophages, was observed in the tissues around the graft valves in all groups (Figure 4A). Herein, the infiltration of these inflammatory cells into the surrounding tissues was scored. On average, the score showed decreased tendency in all treatment groups compared to that of the control group; however, no significant difference was found compared to the result of the control group (Figure 4B).
Figure 4
BHV = bioprosthetic heart valve.
Effects of Entelon150, losartan, and rosuvastatin on inflammatory cell infiltration
Compared to the control group, the Entelon-, losartan-, and rosuvastatin-treated groups displayed less infiltration of inflammatory cells (giant cells, macrophages, heterophils, and lymphocytes) into the tissue surrounding the implanted BHV and within the fibers of the implanted BHV; however, no statistically significant difference was found. An intravenous bovine pericardial implant (asterisk) was detected in the microscopic images. 40× scale bar indicates 50 µm. 200× scale bar indicates 100 µm.
Entelon150®, losartan, and rosuvastatin significantly reduce bone morphogenic protein 2, angiotensin II type 1 receptor, S-100, and alpha smooth muscle actin
The expression ratio of BMP2, AIIR, and S-100 in all experimental groups was significantly decreased compared to that in the control group in the tissue around the implanted BHV (Figure 5). Expression of α-SMA, a myofibroblast marker, did not change in all experimental groups compared to that in the control group. The numerical results are listed in Table 1.
Figure 5
Effects of each drugs on the expression of calcific, inflammatory, and myofibroblastic markers. Data are presented as mean±standard deviation. 400× scale bar indicates 50 µm.
α-SMA = alpha smooth muscle actin; AIIR = angiotensin II type 1 receptor; BMP2 = bone morphogenic protein 2.
Table 1
Summary of the experimental results
Entelon150® reduces bone morphogenic protein 2 and interleukin-6 expression in vascular tissues around the implanted bioprosthetic heart valve
Western blot analysis was performed to determine the expression of BMP2, IL-6, and OPN (Figure 6A). The relative density was obtained by dividing the value of the target protein by that of β-actin, which served as the intrinsic control (Figure 6B). Western blot analysis revealed that the level of BMP2 was significantly reduced by Entelon150® (p=0.031). However, no significant differences were found for the losartan and rosuvastatin groups compared to the control group. IL-6 level showed reduced tendency by Entelon150® and losartan, despite no significant difference relative to the level in control. Further, the rosuvastatin group had a higher level of IL-6 than the Entelon150® group (p=0.012) There was no significant difference in OPN expression between the experimental groups and control group.
Figure 6
Effects of each drug on the calcific and inflammatory protein expression. Data are reported as mean±standard deviation.
BMP2 = bone morphogenic protein 2; IL-6 = interleukin-6; OPN = osteopontin.
DISCUSSION
Grape fruit (Vitis vinifera) is rich in flavonoid compounds, including gallic acid, catechins, epicatechins, ferulic acid, and proanthocyanidin, that exhibit beneficial effects, such as antioxidant, anti-inflammatory, anti-tumor, and anti-aging.10), 11) Entelon150® prevents angiogenesis by disrupting the vascular endothelial growth factor (VEGF)/VEGF receptor signaling and decreases inflammation by reducing IL-6 activity in different disease models. Entelon150® has been proven to have anti-inflammatory and antioxidant effects and is used to treat various diseases.12), 13), 14) In a previous study, Entelon150® was found to reduce calcium content and inflammation in a BHV-implanted dog model.8) In this comparative study using rabbit species to establish the intravascular BHV-implanted model, we found that Entelon150® significantly reduced the calcium content in implanted BHV tissue, reduced inflammatory cell infiltration, and downregulated the expression of IL6, BMP2, S-100, and AIIR in the vascular tissue around the BHV implantation site. These results were either better or similar to that of losartan and rosuvastatin, suggesting the therapeutic efficacy of Entelon150® for the BHV-implanted condition.
Numerous studies have shown that BHVs do not fully address long-term needs as prosthetic valve replacements owing to inevitable structural valve degeneration.1), 2), 15), 16) Herein, dead cells, cell debris, and collagen crosslinks were found around the implanted BHV. Calcium ions and the specific space structure for calcification were structured by dead cell. Consequently, serum protein and lipid infiltration, cytokines, xenoantibodies secreted by B cells, and thrombosis would activate macrophages and induce an inflammatory response, leading to BHV calcification.16) Overall, the limitations of BHV associated with calcification serve as major issues for researchers and surgeons. Accordingly, research on treatment techniques is underway to overcome these drawbacks.
To date, no clinical drugs have been approved for BHV calcification; however, statins have been reported to attenuate BHV calcification.7), 17) According to the pathology related to immune response and BHV calcification, immunosuppressive therapy, including steroid and anti-thymocyte globulin treatment, has been evaluated as a strong candidate for the prevention of BHV calcification.18), 19) In a previous study using a rabbit model, losartan (AIIR blocker) was demonstrated to be effective at attenuating BHV calcification.20) Further, in another study using a dog model, Entelon150® was found to be effective at attenuating BHV calcification and inflammation.8) This result was associated with inflammatory responses caused by IL-6, BMP-2, and OPN.
However, there are currently no studies that directly compare the anti-calcification and anti-inflammatory effects of each drug on BHV using a single animal model. In this experiment, we utilized a rabbit model to directly compare the effects of each drug, and they demonstrated significant reduction in BHV calcification and inflammation compared to the control group, similar to the findings from previous research. Particularly, Entelon150®, which we considered as a new alternative candidate, exhibited comparable anti-inflammatory and anti-calcification effects to losartan and rosuvastatin. Furthermore, in addition to the previous dog model, this study further solidifies the potential of Entelon150® as a new alternative that can delay BHV calcification and degeneration, as it showed similar effects in delaying calcification and inflammation of BHV implanted in the rabbit model. These findings emphasize the promising potential of Entelon150® as a new alternative drug for delaying BHV calcification and degeneration.
As shown in Figures 1 and 2, surgical procedures were successfully performed to fix BHV to the inner wall of the jugular vein of New Zealand white rabbit. There were no clinical symptoms in animals administered the drug for 6 weeks and no deaths. The animals were killed and tissue samples were collected for each analysis. Most importantly, the calcium content measurement performed in this study confirmed the anti-calcification effects of all the test drugs. In particular, Entelon150® reduced the calcium content to a greater extent than the other administered drugs (Figure 3). Inflammatory cell infiltration of vascular tissue was confirmed (Figure 4). According to the pathophysiology of BHV calcification,16) infiltration of inflammatory cells, such as fibroblasts and macrophages, was observed around the graft valve in all groups. Unfortunately, no significant difference was found between the drug-treated and control groups; however, the histopathological score for inflammatory cell infiltration was decreased in the Entelon150® group compared to that for the control and other experimental groups. Additionally, we confirmed the expression of calcification, inflammation, and myofibroblastic markers using IHC (Figure 5). BMP2 is a member of the transforming growth factor superfamily and exerts conventional and ectopic osteogenesis.21) S-100 is a family of calcium-binding proteins that regulates calcium balance.22) Recently, S-100 was found to regulate macrophage inflammation.22) AIIR is a receptor of angiotensin-2 and is known to stimulate inflammation and macrophage cholesterol accumulation. Further, BMP2 expression in valvular fibroblasts can lead to calcium deposition.23) α-SMA is a tissue myofibroblast marker that initiates calcification, including the infiltration of inflammatory cells and lipid accumulation.24) As shown in Figure 5B, the expression ratio of BMP2, S-100, and AIIR was significantly reduced in all experiment groups compared with that in the control group. However, α-SMA expression did not change in any group compared with that in the control group. The results of BMP2, AIIR, and α-SMA in the group treated with Entelon150® or losartan were consistent with those of our previous studies.8), 20) The protein expression of BMP2, IL-6, and OPN was determined in the BHV-implanted tissue (Figure 6). As expected, BMP2 expression was significantly decreased in the Entelon150® group, whereas no significant results were observed for other drugs. IL-6, an inflammatory cytokine induced by BHV degeneration, was not significantly reduced in any experimental group. Rosuvastatin significantly increased IL-6 expression compared with that found in the control group. Expression of OPN, a bone genesis marker, did not change in any of the experimental groups compared with that in the control group. Overall, Entelon150®, losartan, and rosuvastatin were found to have a positive effect on BHV degeneration. However, slightly better results were obtained in the Entelon150® group relative to the other groups. In addition, in this study, we employed clinically acceptable human doses of each drug (Entelon150® 300 mg/day, losartan 100 mg/day, and rosuvastatin 20 mg/day) according to the body surface area of the rabbit (Entelon150® 15.4 mg/kg/day, losartan 5.14 mg/kg/day, and rosuvastatin 1 mg/kg/day). From a clinical perspective, this dose is markedly more realistic than the drug dose applied in previous studies (Entelon150® 300 mg/day in a dog model, losartan 25 mg/kg/day in a rabbit model, and rosuvastatin 20 mg/kg/day in a rat model).7), 8), 20)
Adjuvant drugs may play an important role in the prognosis of BHV implantation. During long periods of drug intake, attention should be paid to the resulting side effects. Losartan has been reported to cause side effects, such as neuropsychiatric and hepatic abnormalities, sexual dysfunction, and angioedema.25) Rosuvastatin can also cause side effects, such as myalgia, myopathy, serum transaminase elevation, and renal failure.26) Entelon150® is an extract of natural food. As a result, no particular side effects have been identified to date. Further, Entelon150® has not been recognized to be burdensome after long-term use. In this study, Entelon150® had comparable or superior effects on BHV calcification and inflammation compared with losartan or rosuvastatin.
This study had several limitations. First, 28 healthy rabbits were employed in this study; however, each group only contained 7 rabbits, which is too small to generalize the experimental results. Furthermore, during the experiment period, we monitored the health status of the rabbits, including their body weight, on a daily basis. However, we did not evaluate the baseline immune and inflammatory status of each subject before the experiment. It is possible that tissue responses may vary depending on the baseline condition of each subject. However, we did not take this factor into consideration in this experiment. Second, the experimental results and responses in humans may differ. Valve tissue was transplanted into the jugular vein; the response to this transplantation and BHV implantation in the arterial position may also differ. For intra-arterial implantation, a technical limitation was identified as the carotid artery was too small (2 mm). Although both intravenous implantation and intra-arterial implantation involve direct blood contact, there is a difference in the presence of arterial pulse with intra-arterial implantation.27) However, previous comparative experiment has shown that arterial pressure during intra-arterial implantation did not have an impact on tissue calcification.27) In addition, in a previous study, we verified that the intravenous implantation model had the highest degree of BHV calcification compared to other implantation models, highlighting that it is the most appropriate model for studies on BHV calcification.3), 27) Thirdly, we did not investigate the differences based on the dosage of each drug. The differences in efficacy based on the dose of each drug are an important issue to be addressed in future research. Further studies will be necessary to understand the effects of the drugs in relation to their dosage. Lastly, the degeneration of BHVs progresses over a period of ten years or more. However, in order to compare our results with previous studies, we chose a duration of 6 weeks.3), 8), 20) Additionally, considering the experimental period and the associated costs and manpower, we decided to maintain the treatment period at 6 weeks. And we did not consider the presence of full intimalization after 6 weeks of BHV implantation. Since our experimental model observed only a 6-week period, further research is necessary to investigate the effects of drugs over longer periods of several years, as seen in real-life situations.
Overall, in this comparative study using a rabbit model, Entelon150® exhibited the significant effect, ultimately reducing calcification and inflammation in the intravascular bovine pericardium. Therefore, Entelon150® could be a promising therapeutic alternative for reducing BHV inflammation and calcification.
Funding:This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2021R1G1A101339812).
Conflict of Interest:Hanlim Pharmaceutical provided partial funding for this study, which may present a potential conflict of interest. However, the company was not involved in the design or analysis of this research and did not have access to this article before its submission.
Data Sharing Statement:The data generated in this study is available from the corresponding authors upon reasonable request.
Author Contributions:
Conceptualization: Shin HJ.
Data curation: Seo J, Kim S.
Formal analysis: Seo J, Kim S.
Funding acquisition: Shin HJ.
Investigation: Seo J, Kim S.
Methodology: Seo J, Kim S.
Supervision: Shin HJ.
Validation: Lee JS, Rahman MM, Shin HJ.
Visualization: Kim S.
Writing - original draft: Lee JS.
Writing - review & editing: Rahman MM, Shin HJ.
References
-
Kim DH, Park HK, Park YH, et al. Degenerative calcification of pericardial bioprostheses: comparison of five implantation methods in a rabbit model. J Heart Valve Dis 2015;24:621–628.
-
-
Côté N, Couture C, Pibarot P, Després JP, Mathieu P. Angiotensin receptor blockers are associated with a lower remodelling score of stenotic aortic valves. Eur J Clin Invest 2011;41:1172–1179.
-
-
Eishi K, Ishibashi-Ueda H, Nakano K, et al. Calcific degeneration of bioprosthetic aortic valves in patients receiving steroid therapy. J Heart Valve Dis 1996;5:668–672.
-
-
O’Brien KD. Pathogenesis of calcific aortic valve disease: a disease process comes of age (and a good deal more). Arterioscler Thromb Vasc Biol 2006;26:1721–1728.
-
-
Meuris B, Ozaki S, Herijgers P, Verbeken E, Flameng W. Bioprosthetic tissue calcification: influence of blood contact and arterial pressure. an experimental study in rats and sheep. J Heart Valve Dis 2003;12:392–399.
-
Cited by
MeSH Terms
Figures
1 / 6
Tables
1 / 1
Funding Information
-
National Research Foundation of Korea
2021R1G1A101339812
