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

Objective Inflammation and calcification are major factors responsible for failure 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 significantly 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 46 bioprosthetic heart valve leaflets with specific treatment to increase its longevity. Heart valves 47 made of a bovine pericardium are safe, offer improved hemodynamics, have less risk of 48 thrombosis, and do not need long-term anticoagulant therapy [1,2]. However, the durability of 49 the bovine pericardium is a major problem, as it is prone to valve calcification, structural  Interleukin-6 (IL-6) plays an important role in increasing bone morphogenic protein 2/4 (BMP-61 2/4) expression in vessels and valve tissue, thereby leading to vascular calcification [3]. It has 62 been shown that application of GSE reduces IL-6 activity in different disease models [10,11]. 63 To the best of our knowledge, there have been no studies examining the effects of GSE on 64 bovine pericardium implants. It is thus unknown whether GSE could prevent bovine 65 pericardium calcification and degeneration. Therefore, the purpose of this study was to 66 investigate the anti-inflammatory and anti-calcification effects of Entelon150 ® on intravenous 67 bovine pericardium implants in beagle dogs.  tubes. An 8-mm longitudinal incision was then made in the jugular vein using a no. 11 surgical 93 blade. A commercially available bovine pericardium (PERIBORN ® Bovine Pericardium, 94 BP0506, Taewoong Medical Co., Ltd. Gimpo-si, South Korea) was used in this study. Before 95 implantation, the Bovine pericardium was rinsed for 30 min in 500 ml of sterile physiological 96 saline. The antibiotic cephradine (30 mg/kg i.v, bid) and the analgesic tramadol (2 mg/kg i.v., 97 tid) were injected for three days after surgery. An approximately 3-mm rectangular 98 bioprosthetic was fixed to the inner wall of the jugular vein using a 6-0 polypropylene running 99 suture. The jugular vein was closed by angioplasty using a 6-0 polypropylene running suture.

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The bovine pericardium was also implanted into the right external jugular vein as described

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There were no differences in body weight between the control and Entelon150 ® -treated 168 groups (Fig 2 A). Vascular patency was evaluated using ultrasonography and CT scanning.

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No interruption of flow patency was observed on either CT scans (Fig 2, B and C) or 170 ultrasonographs (Fig 3).

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Calcified lesions were not observed around the vessel or in the implants in either control or 172 Entelon150 ® -treated groups. The Ca 2+ level in the Entelon150 ® -treated group (0.56±0.14 173 mg/g) was significantly lower than that in the NC group (1.48±0.57 mg/g) (P < 0.05, Fig 4).

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However, the expression of OPN was not significantly different between two groups (Fig 5).

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Histopathological examination revealed infiltration of chronic inflammatory cells such as 180 fibroblasts and macrophages around the graft in all groups. However, the inflammation level 181 of the Entelon150 ® -treated group (1.50±0.58%) was significantly lower (P < 0.001) than the 182 NC group (2.25±0.96%). In particular, a basophilic substance presumed to be the earliest sign 183 of calcium deposition was observed in the NC group between the intercellular matrixes of the 184 peri-graft tissue (Fig 6). Immunohistochemical staining revealed that BMP-2 levels in the 185 Entelon150 ® -treated group (1.27±0.06%) were significantly (P < 0.05) lower than those in 186 the NC group (1.67±0.31%). However, the expression of α-SMA was not significantly 9 187 different between the two groups (Fig 7).  In addition, we found that bovine pericardium triggered an immunological response, as we 10 210 observed a significant elevation of IL-6 in the NC group. Steroidal anti-inflammatory therapy 211 significantly reduces the incidence of postoperative valve tissue rejection in patients, indicating 212 that suppressing the valve-induced immunological response may improve the postoperative 213 durability of bioprosthetic aortic valve implants [16]. Importantly, our data showed that 214 Entelon150 ® treatment significantly lowered IL-6 levels, thus mitigating inflammation. calcified tissue has also been reported [22,23]. Consistent with these findings, the expression 239 α-SMA in the bovine pericardium was lowered by Entelon150 ® treatment; however, this 240 difference was not statistically significant. 241 We have previously found that the angiotensin II type 1 receptor blocker losartan attenuates 242 bioprosthetic valve leaflet calcification in a rabbit model of intravascular implantation [3].

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Calcification of bovine pericardium appears unrelated to certain mechanisms, but rather 244 appears related to reduced inflammation and substances like IL-6, BMP-2, and OPN. Any 245 substance that lowers inflammation through IL-6, BMP-2, and OPN may help prevent 246 calcification. From this point of view, Entelon150 ® , which consists of grape seed extract, will 247 be more powerful than other synthetic medications at preventing calcification. humans. Furthermore, we reported that the calcium content was higher in intravenous implants 256 than in arterial patch implants [24]. We performed our experiments in beagle dogs rather than 257 rabbits; however, we think our results are consistent with the findings in our rabbit intravascular