Comparative Investigation of the Effects of Entelon150®, Losartan, and Rosuvastatin Following Subdermal Valve Prosthesis in a Rat Model

TL;DR

This study compares the effects of three drugs on reducing calcification and inflammation in heart valve implants in rats.

Contribution

The study is the first to compare Entelon150®, losartan, and rosuvastatin in a rat model of bioprosthetic heart valves.

Findings

All three drugs reduced calcium content in heart valve leaflets compared to the control group.

Entelon150® showed the greatest reduction in inflammatory markers IL-6, OPN, and BMP-2.

Losartan was most effective in reducing inflammatory cell infiltration.

Abstract

Background/Objectives: 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). This study aimed to compare the anti-calcification and anti-inflammatory effects of each drug in a rat model. Methods: Twenty-eight female Sprague-Dawley rats (two weeks old) were implanted with BHV leaflets in the dorsal subcutis. They were divided into control, losartan (10.3 mg/kg/day), rosuvastatin (2 mg/kg/day), and Entelon150® (30.8 mg/kg/day) groups. Eight weeks later, the calcium level and inflammatory cell infiltration in the BHV leaflets as well as the expression levels of IL-6, osteopontin, and BMP-2 in the surrounding tissues were measured. Results: Losartan-, Entelon150®-, and rosuvastatin-treated groups showed a decrease in the calcium content as compared to the control group in the order. A reduction in the inflammatory cell infiltration was observed in the treatment groups (losartan > Entelon150® > rosuvastatin) as compared to that in the control group. The treatment groups showed a decrease in IL-6, OPN, and BMP-2 expression levels as compared to the control group in the following order: Entelon150® > losartan > rosuvastatin. Conclusions: The calcification of the BHV leaflets and inflammation of the surrounding tissues in treatment groups were lower than those in the control group and were comparable to each other. Entelon150® showed comparable anti-inflammatory and anti-calcification effects and may represent a potential therapeutic candidate for prolonging BHV durability, although further validation is required.

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1. Introduction

Bioprosthetic heart valves (BHVs) have been widely used since their development in the 1960s [1]. BHV has the advantage of a lower risk of thromboembolism compared to mechanical valves and does not require the use of anticoagulants, but its durability is less than that of mechanical valves due to degeneration over time, and the probability of reoperation is high [2]. The deterioration of BHV is mainly caused by calcification, immune response, and mechanical degeneration [3,4].

Several studies have been conducted to identify treatments to prevent or delay BHV deterioration [4,5,6]. Previous studies have reported that losartan (angiotensin II type 1 receptor blocker) and rosuvastatin (HMG-CoA reductase inhibitors) attenuate BHV calcification [5,6,7]. In addition, Entelon150^®^ (Vitis vinifera seed extract), a natural herbal preparation, has been shown to be effective in reducing calcification and inflammation of the bovine pericardium [8]. Since these studies have been conducted separately using different animal models, the effects of the drugs on BHV cannot be compared.

Therefore, the aim of this study was to compare the anti-calcification and anti-inflammatory effects of losartan, rosuvastatin, and Entelon150^®^ in a rat model.

2. Materials and Methods

2.1. Animals, Diets, and Experimental Design

All animal experiments were approved by the Institutional Animal Care and Use Committee of Korea University Medical Center (approval no. KOREA-2021-0219). Twenty-eight healthy female Sprague-Dawley (SD) rats (two weeks old; mean body weight of 50 g after 1 week of quarantine) were purchased from Orient Bio Inc. (Seongnam, Gyeonggi-do, Republic of Korea). The sample size of seven animals per group was determined in reference to previously published studies that utilized similar rat or rabbit models for bioprosthetic valve research [6,7,8]. They were housed separately in stainless steel cages (895 mm W × 795 mm L × 765 mm H) in an environmentally controlled room (temperature: 23 ± 3 °C, relative humidity: 55 ± 15%, ventilation frequency: 10–20 times/h, light cycle: 8 a.m. to 8 p.m., and illumination: 150–300 Lux). Food and sterilized water were available ad libitum. After 1 week of quarantine, all SD rats were implanted with BHV into the dorsal subcutis. To observe the effects of losartan, rosuvastatin, and Entelon150^®^, the implanted SD rats were divided into control (fed a normal diet, n = 7), losartan (fed a diet including 400 mg losartan/3 kg diet, n = 7), rosuvastatin (fed a diet including 75 mg rosuvastatin/3 kg diet, n = 7), and Entelon150^®^ (fed a diet including 1100 mg Entelon150^®^/3 kg diet, n = 7) groups. Eight weeks later, body weight of SD rats and Ca^2+^ levels in implanted BHVs were measured.

2.2. Specialty Diet Preparation

Specialized diets containing losartan, rosuvastatin, or Entelon150 ^®^ were prepared to deliver drugs into SD rats through a non-invasive and non-stressful procedure. The dose of each drug was determined according to the animal equivalent dose (AED) [9]. For a 60 kg-human, the doses of losartan, rosuvastatin, and Entelon150^®^ are 100, 20, and 300 mg/day, respectively. The Km factor for humans and rats are 37 and 6, respectively. The amounts of drugs required to prepare specialty diets were determined using the following equation:

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The concentrations of losartan, rosuvastatin, and Entelon150^®^ in the specialty diets were 10.3 mg/kg/day, 2 mg/kg/day, and 30.8 mg/kg/day, respectively. To feed the exact amounts of drugs to SD rats, it was assumed that the amount of diet consumed by an adult SD rat was 15 g. Further, it was assumed that the weight of female SD rats used in the experiment increased by 20 g per week from 50 g (three weeks old) and the amount of feed consumed increased with the rat’s age. The dosages of the drugs were derived from those required for 8-week-old rats (170 g). Losartan (350 mg), rosuvastatin (68 mg), or Entelon150^®^ (1.0472 g) were mixed with 3-kg regular diet. Diets containing drugs were then sterilized by gamma-ray irradiation.

2.3. Animal Experiment

Each SD rat was anesthetized with an intravenous injection of alfaxalone (10 mg/kg) and xylazine (5 mg/kg). Commercially available bovine pericardial BHVs (Carpentier-Edwards Perimount Magna pericardial prosthesis; Edwards Lifesciences, Irvine, CA, USA) were used in the study. BHV leaflets were surgically implanted into the dorsal subcutis after incision of the dorsal midline skin and fixed with a 6-0 polypropylene suture. And the skin was sutured with a simple interrupted pattern using a 4-0 polypropylene suture. Three days after surgery, 5 mg/kg enrofloxacin and 5 mg/kg ketoprofen were injected subcutaneously for infection prophylaxis and analgesia, respectively. The implanted SD rats were randomly allocated into four groups (control, losartan, rosuvastatin, and Entelon150^®^ groups) and fed either the regular (control group) or drug-containing diet (treatment groups) for 8 weeks.

2.4. Quantitative Determination of Calcium

Eight weeks after BHV implantation, the animals were euthanized by CO_2_ inhalation, and implanted BHV samples were collected. After thoroughly removing the surrounding connective tissues, the samples were frozen for 24 h at −80 °C. Moisture was removed from the samples using a freeze-dryer for 24 h, and then the samples were weighed and placed in 5 mL of aqua regia in a 100-mL beaker. The beaker was slowly heated on a hot plate for 6 h at 70–80 °C to avoid tissue disruption from rapid thawing and rewarming, followed by heating for 5 h at 140–150 °C; 5 mL of H_2_O_2_ was then added. To ensure an objective evaluation, the calcium content of the samples was analyzed by an external professional institution blinded to the treatment groups using an inductively coupled plasma atomic emission spectrometer (ICP-AES, PerkinElmer, Norwalk, CT, USA).

2.5. Immunoblotting

To ensure an objective evaluation, all subsequent protein extraction and immunoblotting procedures were performed by an external professional institution blinded to the treatment groups. To investigate the expression level of IL-6, osteopontin, and BMP-2, the samples were stored at −70 °C. Proteins were extracted with 400 µL of PRO-PREP protein extraction solution (Intron Biotechnology, Seongnam, Kyungki-Do, Republic of Korea), followed by tissue homogenization using a tube homogenizer (Daihan Scientific, Wonju, Republic of Korea). After 30 min on ice, samples were centrifuged at 13,000 rpm for 15 min at 4 °C. The supernatant was collected. Protein content was quantified using a BCA protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). For immunoblotting, 50 mg of total protein extract was separated using 15% SDS-PAGE (70 V, 4 h) and transferred to a nitrocellulose (NC) membrane (50 mA, 10 h) using a Trans Blot SD semi-dry transfer cell (Bio-Rad, Hercules, CA, USA). After blocking with 5% BSA for 1 h, the blotted membranes were incubated with anti-BMP-2 antibody (ab6285, 1:1000 dilution, Abcam, Cambridge, UK), anti-osteopontin (ab63856, 1:1000 dilution, Abcam), and anti-IL-6 antibody (ab6672, 1:1000 dilution, Abcam) antibodies in TBS-T with 5% BSA. β-actin detected using anti-β-actin antibody (SC-47778, 1:2000 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. After incubation with the primary antibodies, the membranes were washed with TBS-T buffer three times and then incubated with secondary goat anti-rabbit immunoglobulin G horseradish peroxidase (ab6789, 1:2000 dilution, Abcam) for the anti-BMP-2 antibody, anti-osteopontin, and anti-IL6 antibodies. Anti-β-actin antibody was directly conjugated with horseradish peroxidase. Proteins were detected using an enhanced chemiluminescence detection reagent (Intron Biotechnology, Kyungki-Do, Republic of Korea); the blotted membranes were imaged using a Chemidoc Touch Imaging System (Bio-Rad) for immunoblot analysis.

2.6. Histopathologic Evaluation

For histological analysis, the implanted BHV leaflets were fixed in 10% neutralized buffered formalin for 10 days and embedded in paraffin using standard methods. Sections 4-µm thick were stained with hematoxylin and eosin to count the hematoxylin-stained cells. To quantify inflammatory cell infiltration, hematoxylin-stained nuclei of inflammatory cells were counted in five randomly selected high-power fields (HPF, ×400 magnification) per slide. The analysis was performed by an examiner blinded to the treatment groups to ensure objectivity.

2.7. Statistical Analysis

All data are expressed as mean ± standard error. Statistical analyses were performed using IBM SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA). Differences among the four experimental groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test for multiple comparisons. Additionally, 95% confidence intervals (CIs) and effect sizes (partial eta-squared, η^2^) were reported to enhance the robustness and transparency of the statistical conclusions. A p value ≤ 0.05 was considered statistically significant.

3. Results

3.1. Entelon150® and Rosuvastatin Significantly Reduced the Calcification of Implanted BHV Leaflets

Figure 1 shows the mean body weight of SD rats in the control and treatment groups eight weeks after BHV implantation. No treatment groups showed significant difference in the mean body weight compared with that of the control group. We investigated the effect of each drug on calcium precipitation in retrieved BHV leaflets. Calcium levels in retrieved BHV leaflets were quantified using ICP-AES. As shown in Figure 2, the mean calcium level in retrieved BHV leaflets in each treatment group was lower than that in the control group. The mean calcium levels were 0.718 ± 0.061 mg/g (Control), 0.209 ± 0.065 mg/g (Losartan), 0.479 ± 0.012 mg/g (Entelon150^®^), and 0.505 ± 0.050 mg/g (Rosuvastatin). One-way ANOVA with Tukey’s post hoc test revealed that the mean calcium levels in all treatment groups—Losartan (p < 0.001), Entelon150^®^ (p = 0.015), and Rosuvastatin (p = 0.035)—were significantly lower than those in the control group. The 95% CIs for the mean differences compared to the control group were 0.276–0.742 mg/g for Losartan, 0.006–0.472 mg/g for Entelon150^®^, and 0.020–0.446 mg/g for Rosuvastatin. The large effect sizes (η^2^ > 0.70) across all markers confirmed the substantial impact of these treatments.

3.2. Entelon150®, Losartan, and Rosuvastatin Significantly Reduced the Inflammatory Cell Infiltration in Implanted BHV Leaflets

Paraffin blocks were used to investigate the extent of inflammatory cell infiltration in the implanted BHV leaflets. As shown in Figure 3, One-way ANOVA revealed that inflammatory cell infiltration in all treatment groups was significantly lower than that in the control group (p < 0.001). The 95% CIs for the mean differences in cell counts compared to the control group were 118.5–293.4 cells/field for Entelon150^®^, 139.0–313.9 cells/field for Losartan, and 91.0–265.9 cells/field for Rosuvastatin. Notably, Entelon150^®^ demonstrated a potent anti-inflammatory effect, with cell counts markedly lower than the control group, comparable to the Losartan and Rosuvastatin groups. High effect sizes were also observed for the reduction in inflammatory cell counts (η^2^ > 0.70).

3.3. Entelon150® Considerably Reduces the Expression Level of Osteopontin, IL-6, and BMP-2

Figure 4 shows the expression level of OPN, IL-6, and BMP-2 in each group in Western blot analysis. The relative density was determined by dividing the band density of the target protein by that of β-actin, which served as an intrinsic control. The expression levels of osteopontin, IL-6, and BMP-2 in all treatment groups were significantly lower than those in the control group (p < 0.05). Notably, the Entelon150^®^ group showed the most potent inhibitory effect, reducing markers to 40.3%, 21.1%, and 12.9% of the control values, respectively (p < 0.001).

4. Discussion

This is the first study to compare the anti-calcification and anti-inflammatory effects of Entelon150^®^, losartan, and rosuvastatin on BHV implants by subcutaneous implantation of BHV leaflets in SD rats. After feeding Entelon150^®^ (30.8 mg/kg/day), losartan (10.3 mg/kg/day), and rosuvastatin (2 mg/kg/day) for eight weeks, calcium levels in retrieved BHV leaflets in each group were measured. The degree of inflammatory cell infiltration in BHV leaflets was evaluated. As compared to the control group, calcium levels in treatment groups showed a decrease in the order of losartan, Entelon150^®^, and rosuvastatin. Inflammatory cell infiltration was decreased in the order of losartan, Entelon150^®^, and rosuvastatin. The expression levels of IL-6, osteopontin, and BMP-2 decreased in the order of Entelon150^®^, losartan, and rosuvastatin. The expression levels of IL-6, osteopontin, and BMP-2 in the Entelon150^®^ group showed the most significant decrease. BMP-2, a member of the transforming growth factor superfamily, plays an important role in inflammation and vascular calcification [10]. An elevated level of IL-6, which is an inflammatory marker, is associated with adverse cardiovascular events, including coronary artery calcification, and is known to be a predictor of death [8,11]. Osteopontin is a secreted matricellular cytokine whose level increases in the presence of inflammatory conditions and vascular calcification [12,13]. The degree of calcification of the BHV leaflets and inflammation of the surrounding tissues in each treatment group was significantly lower than that of the control group (Central illustration).

Serum protein and lipid infiltration, cytokines, xenoantibodies secreted by B cells, and thrombosis activate macrophages and induce an inflammatory response, resulting in BHV calcification [14]. Immune and inflammatory reactions and the resulting calcification are the main mechanisms of BHV degeneration, and with this in mind, several drugs have been studied for anti-inflammatory and anti-calcification purposes. In experiments using rabbits, it was demonstrated that losartan (angiotensin II type 1 receptor blocker) is effective in reducing BHV calcification [7]. Based on the fact that cholesterol levels are associated with BHV calcification, the anti-calcification effect of rosuvastatin on BHV in SD rats was reported in a previous study [6]. Entelon150^®^ has also been shown to attenuate inflammation and calcification of the bovine pericardium in dogs [8]. All of these drugs are presumed to be somewhat effective in delaying the degeneration of BHV; however, to date, there are no studies comparing the effectiveness of these drugs.

Grapes (Vitis vinifera), the source of Entelon150^®^, are rich in flavonoid compounds, including gallic acid, catechin, epicatechin, ferulic acid, and proanthocyanidin, which are known to have beneficial effects such as antioxidant, anti-inflammatory, antitumor, and anti-aging [15,16,17]. Entelon150^®^ has been shown to have anti-inflammatory and antioxidant effects in various diseases [18,19]. As a natural substance, Entelon150^®^ is generally well-tolerated and may offer a therapeutic option with fewer adverse events associated with conventional synthetic drugs.

Previous studies using animal models have used the drugs at doses (Entelon150^®^, 300 mg/d in a dog model; losartan, 25 mg/kg/d in a rabbit model; and rosuvastatin, 20 mg/kg/d in a rat model) that were excessively higher than the actual doses of the drugs taken by patients (Entelon150^®^, 300 mg/d; losartan, 100 mg/d; and rosuvastatin, 20 mg/d), which are different from the doses recommended in clinical practice [6,7,8]. In this study, a realistic drug dose suitable for the weight of SD rats was applied based on the actual drug dose used for adults weighing 60 kg (Entelon150^®^, 30.8 mg/kg/d; losartan, 10.3 mg/kg/d; and rosuvastatin, 2 mg/kg/d). The average weight of SD rats in each group was similar even after eight weeks of drug use. The mean calcium level in BHV leaflets in all treatment groups—Losartan (p < 0.001), Entelon150^®^ (p = 0.015), and Rosuvastatin (p = 0.035)—was significantly lower than that in the control group. These results, analyzed via one-way ANOVA and Tukey’s post hoc test (showing large effect sizes, η^2^ > 0.70), demonstrate that Entelon150^®^ exerts a meaningful anti-calcification effect.

Our results indicate distinct therapeutic profiles for the tested agents. Losartan demonstrated the greatest efficacy in reducing calcium content, suggesting a potent direct anti-calcific effect. In contrast, Entelon150^®^ showed the most significant reduction in inflammatory markers, such as IL-6, OPN, and BMP-2 (p < 0.05). Notably, these molecular analyses were conducted on the reactive tissue surrounding the leaflets due to technical constraints; glutaraldehyde-fixed leaflets exhibit extensive protein cross-linking, which makes reliable protein extraction for Western blotting unfeasible. Furthermore, as fixed leaflets are biologically inert, the signals driving calcification originate from the surrounding host tissue via paracrine signaling. Therefore, the significant down-regulation of IL-6 and OPN in the Entelon150^®^ group suggests a potent suppression of macrophage-mediated inflammatory responses, as these markers are closely associated with the recruitment and activation of macrophages in calcified tissues. This suggests that Entelon150^®^’s therapeutic potential lies in targeting these upstream inflammatory pathways, whereas Losartan appears to act more effectively on the calcification endpoint itself.

Entelon150^®^ showed anti-inflammatory effects comparable to those of the other agents, while Losartan exhibited the strongest direct anti-calcification effect. Furthermore, due to its favorable safety profile, it may be a suitable candidate for long-term administration to prolong the lifespan of BHV.

This study has several limitations. First, we could not explain the mechanisms by which each drug exerts its anti-calcification and anti-inflammatory effects. Second, the subcutaneous model lacks key physiological factors such as hemodynamic stress, valve leaflet motion, and direct blood–tissue interactions. Consequently, phenomena such as thrombosis and endothelial cell infiltration–proliferation could not be evaluated. Thus, our results should be viewed as preclinical exploratory findings. Future validation in intravascular models is required to confirm these effects in a clinical context. Third, this study did not identify specific immune cell subtypes (e.g., CD68+ macrophages or CD3+ T cells) through immunohistochemistry. Although general inflammatory cell infiltration was quantified via H&E staining, further studies using specific IHC markers are needed to fully elucidate the distinct roles of different immune cell populations in the protective effects of Entelon150^®^. Fourth, molecular analyses were performed on the surrounding host tissue rather than the valve leaflet matrix itself. While this approach was necessitated by the technical constraints of protein extraction from glutaraldehyde-fixed tissues, direct analysis of the valve matrix in future studies would provide more specific insights into the intra-leaflet process. Finally, since we used SD rats as experimental animals, responses in humans may differ from the results of this study. Although the drug dosages in this study were calculated based on body surface area (BSA) to approximate human therapeutic levels, interspecies differences in pharmacokinetics—such as drug metabolism, absorption rates, and clearance pathways—may still exist.

5. Conclusions

In this comparative study using a rat model, Entelon150^®^, losartan, and rosuvastatin treatments were associated with a reduction in the calcification and inflammatory cell infiltration of implanted BHV leaflets and expression of IL-6, osteopontin, and BMP-2 proteins in surrounding tissues. Entelon150^®^ shows potential as an alternative strategy to prolong BHV lifespan. However, further validation in advanced translational models is required to confirm its clinical efficacy.