Abstract

Background: This study aims to investigate whether an endothelial marker ephrin-B2 exists in pericardial patches during post-angioplasty repair and whether CD34⁺ endothelial progenitor cells are present in the patches after being implanted into the arterial environment.

Methods: Eighteen eight-week-old male Wistar rats were subjected to laparotomy to check the abdominal aorta below renal arteries, to incise it, and then to perform angioplasty by using porcine or bovine pericardial patch. The control group consisted of 18 age- and weight-matched Wistar rats. Angioplasty was conducted by subcutaneously implanting patches in the control group. The patches were taken on one, three, five, and 30 days after surgery and investigated by histological and immunohistochemical assays, immunofluorescent labeling, Western blotting and reverse transcription polymerase chain reaction.

Results: The patches containing collagen were acellular before angioplasty. After implantation of a bovine pericardial patch, the numbers of ephrin-B2⁺ and CD34⁺ cells increased significantly; however, the test results for CD68, alphaactin and von Willebrand factor were negative. There was a monolayer of cells in the inner luminal surface five days after implantation of a porcine pericardial patch. In contrast, ephrin-B2⁺ or CD34⁺ cells did not appear in the control group. On the postoperative 30th day, there were ephrin-B2⁺ and CD34⁺ cells in the two types of patches.

Conclusion: Ephrin-B2⁺ and CD34⁺ cells began to infiltrate pericardial patches soon after implantation. The patches which allow endothelialization during arterial remodeling are potentially applicable to tissue plasty and angioplasty.

Medical materials, as multidisciplinary products of chemistry and biomedical engineering, have been widely used in surgeries,[1] particularly in angioplasty.[2] Synthetic materials, such as vascular stents, artificial blood vessels and vascular patches, have been also applied in modern vascular surgeries.[3] In particular, vascular patches made of polymer materials of biological origin can improve the therapeutic outcomes of surgery and decrease the risk for postoperative stenosis.[4] Being easily operable and highly biocompatible, porcine and bovine pericardial patches reduce bleeding during suturing and allow direct Doppler hemorheological detection[5] and real-time monitoring of postoperative blood flow. Besides, the implanted blood vessels are highly endurable due to satisfactory remodeling and adaptability. In this study, we aimed to investigate whether endothelial marker ephrin-B2 (EFNB2) existed in pericardial patches during post-angioplasty repair and whether CD34⁺ endothelial progenitor cells (EPCs) were present in the patches after being implanted into the arterial environment.

Methods

All the animal experiments were approved by the Institutional Animal Care and Use Committee of Huaihe Hospital of Henan University and were performed in accordance with the principles of the Animal Research: Reporting in vivo Experiments (ARRIVE) guideline.

The overriding principle is expressed as the 3Rs: Replacement, Refinement, Reduction. The documents recommended herein are available in the website of National Centre for 3Rs (http://www.nc3rs.org.uk).

Preoperative preparation
Eighteen eight-week-old male Wistar rats (mean weight: 210±33 g) were selected, continuously anesthetized by isoflurane, and implanted with patches. The flow rates of oxygen and isoflurane during anesthesia were 0.8 L/min and 3 mL/min respectively, and were 0.6 L/min and 1.5 mL/min during surgery, respectively. Porcine and bovine pericardial patches were purchased from the Tissue Regenix (United Kingdom) and Beijing Balance Medical Co., Ltd. (China), respectively.

Surgical methods
Under general anesthesia, the rat limbs were fixed and abdominal hair was completely shaved. The abdominal skin was sterilized with medical alcohol, covered with a sterile towel drape, and cut until the muscle was reached. The intestinal canal was wrapped and pulled rightwards with gauze moistened with saline. The abdominal wall was, then, stretched to expose the posterior peritoneum. The abdominal aorta below renal arteries was isolated, hypodermically injected with heparin, and blocked about 6 mm from the proximal and distal ends with a microvascular occlusion clamp 1 minute later.[6] After the anterior wall of the abdominal aorta was cut with a 3 mm incision and trimmed into an approximate oval one along the blood vessel, on which pericardial patches trimmed into the same shape, it was interruptedly sutured with 10-0 nylon threads. The microvascular occlusion clamp was removed after surgery to recover the blood flow in abdominal aorta and the suture site was compressed with a cotton swab to prevent postoperative bleeding and possible pseudoaneurysms. Subsequently, the abdominal incision was sutured with 5-0 polyester threads and the rats were kept in cages and warmed for rapid resuscitation.[7] Another age- and weight-matched 18 Wistar rats were subcutaneously implanted with patches by using identical methods as the control group.

Postoperative tissue collection
All rats were anesthetized and fixed again using the methods as mentioned above. The skin and muscle in the middle of chest were cut and the anterior wall of the chest was scissored around the rib to expose the heart. Afterwards, the left ventricle was punctured into which about 30 mL of phosphate buffer was perfused after the liver was cut to drain blood, and, then another 20 mL of 10% formalin solution was perfused to fix systemic tissues. After the abdominal cavity was opened, the intestinal canal was removed to expose the peritoneum, and the patched abdominal aorta was scissored, rinsed with normal saline, and stored prior to use.

Histological assay
The collected samples were fixed in 10% formalin, stored in 70% ethanol, fixed in paraffin, and cut into sections. Specifically, the samples stored in 70% ethanol were dehydrated, soaked in a mixture of dimethylbenzene, and ethanol for two hours, and then in dimethylbenzene solution refreshed twice (1.5 heach time). The dehydrated tissue samples were poured into a box containing liquid paraffin and cooled until white blocks appeared, which were then cut into sections and transferred into a 37 ºC water bath with an ink brush. Afterwards, the unfolded sections were put onto glass slides with water wiped, dried in a 40 ºC oven for about one hour, air-dried, subjected to hematoxylin-eosin staining, and studied under an microscope to count cells[8] and to record the mean number.

Immunohistochemical assay
The glass slides with sections were de-paraffinized in dimethylbenzene-dimethylbenzene-absolute ethanol-absolute ethanol-95% ethanol-90% ethanol-80% ethanol-70% ethanol sequentially (10 minutes in each solution). Subsequently, the sections were washed with water, soaked in 3% H₂O₂ for 10 minutes to remove endogenous catalase,[9] re-rinsed with water, boiled in citrate buffer for three minutes, and cooled down to room temperature (the boilingcooling procedure was repeated). The sections were, then, rinsed with water and washed twice with phosphate buffer on which tissues were marked with a marker pen. Afterwards, the sections were blocked with 1:10 diluted serum and incubated at 37 °C for 0.5 hours, from which excessive serum was dried with an absorbent paper. After addition of primary antibodies and overnight incubation in a 4 °C refrigerator, the sections were rinsed three times with phosphate buffer, added secondary antibodies, incubated again in a 37 °C incubator for 0.5 hours, rinsed three times with phosphate buffer, added 1:100 diluted SABC (Sigma, CA, USA), incubated in the 37 °C incubator for 0.5 hours, washed three times with phosphate buffer, and counterstained. The sections were then dehydrated in 70% ethanol-80% ethanol-90% ethanol-95% ethanol-absolute ethanolabsolute ethanol-dimethylbenzene-dimethylbenzene (2 minutes in each solution), sealed by dropping vegetable glue, and air-dried.

Immunofluorescent labeling
The glass slides with sections were de-paraffinized in dimethylbenzene-dimethylbenzene-absolute ethanol-absolute ethanol-95% ethanol-90% ethanol-80% ethanol-70% ethanol sequentially (10 minutes in each solution). The sections were, then, washed with water, soaked in 3% H₂O₂ for 10 min, re-rinsed with water, boiled in citrate buffer for three minutes, and cooled down to room temperature (the boiling-cooling procedure was repeated). Subsequently, the sections were rinsed with water and washed twice with phosphate buffer, on which tissues were marked with a marker pen. Afterwards, the sections were blocked with 1:10 diluted serum and incubated at 37 °C for 0.5 hours, from which excessive serum was dried with absorbent paper. Anti-EFNB2, CD31 and CD34 antibodies (1:100 diluted), as primary antibodies, were added into the sections which were, thereafter, incubated overnight at 4 °C in the refrigerator. On the next day, the primary antibodies were discarded, and the sections were rinsed three times with phosphate buffer in a constant-temperature shaker (5 minutes each time), added 1:5000 diluted secondary antibodies in dark (fluorescently labeled antibodies corresponding to the primary ones), and incubated in a 37 °C incubator for one hour in dark. After the secondary antibodies were discarded, DAPI was added onto the marked region, and the sections were sealed with nail polish, and stored overnight in a dark plastic box in a 4 °C refrigerator.[10] Fluorescences were observed in dark under a fluorescence microscope using varying wavelengths.

Western blotting
The thoracic aorta, pericardial patches, and inferior vena cava samples were freeze-dried in liquid nitrogen, ground into powders, evaporated, and mixed with protease inhibitor-containing buffer for 30 seconds, the protein concentrations in which were measured with a UV-vis spectrophotometer. The samples were, then, diluted based on proteins with the same masses, homogenized on an ultrasonic vibrator at 4 °C for one hour to extract and to collect the proteins. Proteins with the same quantities were subjected to electrophoresis and membrane transfer. Anti-EFNB2, CD34, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and heat shock protein 90 (HSP-90) antibodies were used as primary antibodies. Protein signals were detected by Pierce ECL reagent (Western Blotting Kit. Thermo Fisher Scientific, MA, USA).[11]

Reverse transcription polymerase chain reaction (PCR)
Ribonucleic acid (RNA) was extracted with Trizol solution (Thermo Fisher Scientific, MA, USA), purified and quantified with an RNeasy Mini kit (Qiagen GmbH, Hilden, Germany), and prepared into cDNA. Real-time fluorescent quantitative PCR was performed by using SYBR GreenSupermix as the labeling group. Glyceraldehyde-3-phosphate dehydrogenase was used as an internal control.

Statistical analysis
Statistical analysis was performed using PASW version 17.0 software (SPSS Inc., Chicago, IL, USA). The differences between two groups and those among multiple groups were compared by t test and one-way analysis of variance (ANOVA), respectively. A p value of <0.05 was considered statistically significant.

Results

Cell counting
The pericardial patches containing collagen were acellular before angioplasty of the abdominal aorta. After implantation of bovine pericardial patches, the numbers of EFNB2⁺ and CD34⁺ cells increased significantly (p<0.05) in a time-dependent manner in the gaps between collagen fibers. Although the majority of the cells migrated toward the patch middle over time, only a few cells appeared around the subcutaneously implanted patches and remained thereafter (Table 1).

Table 1: Cell counts at different time intervals

Cell changes in bovine pericardial patches
Cells were observed in bovine pericardial patches on the postoperative one, three, and five days; however, the test results for CD68, alpha (a)-actin and von Willebrand factor (vWF) were negative. Since immunofluorescent staining using anti-CD31 antibody on the three days did not produce positive results, endothelial cells did not exist. On the postoperative seventh day, CD68⁺, a-actin⁺ and vWF⁺ cells became detectable. In addition, the numbers of EFNB2⁺ and CD34⁺ cells, as suggested by immunofluorescent staining, increased timedependently and moved toward the patch center with a large overlapping area. In contrast, no EFNB2⁺or CD34+ cells were detected in the control group.

To further verify the results, the patches were taken out, from which RNA was extracted for reverse transcription PCR that showed the levels of EFNB2 and CD34 increased significantly after angioplasty (p<0.05) (Figure 1). However, the two genes were not expressed in the control group. Similarly, Western blotting demonstrated EFNB2 and CD34 proteins in the patches (Figure 2), but not in the subcutaneously implanted ones.

Figure 1: EFNB2 and CD34 mRNA expression levels in bovine pericardial patches. mRNA: Messenger ribonucleic acid.

Figure 2: EFNB2 and CD34 protein expression levels in bovine pericardial patches. GAPDH: Glyceraldehyde-3- phosphate dehydrogenase.

Cell changes in porcine pericardial patches
On the postoperative fifth day, dual EFNB2⁺ and CD34⁺ cells also appeared in the porcine pericardial patches. In addition, there were EFNB2 and CD34 proteins in the patches (Figure 3).

Figure 3: EFNB2 and CD34 protein expression levels in porcine pericardial patches. HSP-90: Heat shock protein 90.

Comparison between porcine and bovine pericardial patches
There was a thicker layer of neointima 30 days after implantation of porcine pericardial patch than that after bovine patch implantation (Figure 4). Immunofluorescence assays of both patches showed that CD34+ cells increased over time and tended to accumulate in the center (Figure 5). Similarly, EFNB2⁺ cells, which also increased with an elapsed time, were distributed in the same region as that of CD34⁺ ones. Furthermore, EFNB2 and CD34 proteins were detected in both types of patches (Figures 6 and 7).

Figure 4: Neointimal thicknesses of porcine and bovine pericardial patches on the postoperative 30^th day.

Figure 5: Immunofluorescence assay results.

Figure 6: Protein expression levels in bovine pericardial patches detected by Western blotting. HSP-90: Heat shock protein 90.

Figure 7: Protein expression levels in porcine pericardial patches detected by Western blotting. HSP-90: Heat shock protein 90.

Discussion

In vascular implantation, the shear stress of implants and the migration of cellular constituents during the blood flow should be considered.[12] In particular, endovascular endothelial cells with an intact function and morphology can prevent thrombosis and maintain normal function of the implants.[13] As EFNB2 does not increase along with arterial blood flow dynamics in intravenously implanted endothelial cells, time is needed for the implants to adapt to the specific arterial environment. In our study, five days after implantation of pericardial patches, positive cells began to infiltrate,[14] and EFNB2 and CD34, but not CD68, vWF or a-actin, were expressed. Thus, the patches were found to be adaptable to the arterial environment within a short time after surgery.[15] Also, markers in the arterial endothelial cells originated from initial cells instead of adjoining vascular endothelial cells. On the postoperative 30th day, both porcine and bovine patches underwent endothelialization in the vascular lumen.

Currently, EFNB2 and its receptor EPHB4, which are expressed in arteries and veins, respectively, are commonly used as the corresponding markers.[16] Edema factor protein and EPh receptor-interacting protein, as ligands and receptors,[17] predominantly regulate blood and lymphatic vascular remodeling and endothelial cells, and support cells and smooth muscle cells.[18] It has been previously reported that, unlike intravenous implants, pericardial patches can acquire an arterial marker EFNB2, thereby, being accommodated to the arterial environment through cellular infiltration.[19]

On the other hand, EPCs play a crucial role in vascular remodeling. Under ischemic conditions, EPCs can promote revascularization and repair vascular intima.[20] Therefore, they are potentially eligible for treating lower limb ischemic diseases.[21] In this study, we used an EPC-specific marker,[22] i.e. CD34, to find out whether there were EPCs in the patches.

On the other hand, neither EFNB2⁺ or CD34⁺ cells were detected in the subcutaneously implanted patches, probably as they failed to contact with blood flow in the arterial circulatory system.[23]

Although the formation of luminal endothelial cells in porcine patches preceded that in bovine ones, the latter had neointima within 30 days after surgery. In other words, neointimal endothelialization was both allowed, which reduced the risk of thrombosis, infection and pseudoaneurysm,[24] and augmented the patency rate after angioplasty.

In conclusion, implantation of pericardial patches after angioplasty promoted endothelialization, vascular remodeling, and adaptation to the arterial environment.[25] The patches had EFNB2⁺ and CD34⁺ cells which were prone to differentiation into arterial endothelial cells, which are potentially eligible materials for vascular tissue engineering.

Declaration of conflicting interests
The authors declared no conflicts of interest with respect to the authorship and/or publication of this article.

Funding
The authors received no financial support for the research and/or authorship of this article.

References

1) Langer R, Tirrell DA. Designing materials for biology and medicine. Nature 2004;428:487-92.

2) Jaganathan SK, Supriyanto E, Murugesan S, Balaji A, Asokan MK. Biomaterials in cardiovascular research: applications and clinical implications. Biomed Res Int 2014;2014:459465.

3) Muto A, Nishibe T, Dardik H, Dardik A. Patches for carotid artery endarterectomy: current materials and prospects. J Vasc Surg 2009;50:206-13.

4) Williams C, Xie AW, Emani S, Yamato M, Okano T, Emani SM, et al. A comparison of human smooth muscle and mesenchymal stem cells as potential cell sources for tissue-engineered vascular patches. Tissue Eng Part A 2012;18:986-98.

5) Park JW, Leithäuser B, Rittger H, Brachmann J. Treatment strategies for prevention of cardioembolic stroke in atrial fibrillation. Clin Hemorheol Microcirc 2010;46:251-64.

6) Rustinart GJ. Multiple aneurysms of the right coronary artery; death from a ruptured aneurysm of the abdominal aorta. J Am Med Assoc 1952;149:1129-31.

7) Li X, Guo Y, Ziegler KR, Model LS, Eghbalieh SD, Brenes RA, et al. Current usage and future directions for the bovine pericardial patch. Ann Vasc Surg 2011;25:561-8.

8) Goldman KA, Su WT, Riles TS, Adelman MA, Landis R. A comparative study of saphenous vein, internal jugular vein, and knitted Dacron patches for carotid artery endarterectomy. Ann Vasc Surg 1995;9:71-9.

9) Li X, Jadlowiec C, Guo Y, Protack CD, Ziegler KR, Lv W, et al. Pericardial patch angioplasty heals via an Ephrin-B2 and CD34 positive cell mediated mechanism. PLoS One 2012;7:38844.

10) Luo J, Korossis SA, Wilshaw SP, Jennings LM, Fisher J, Ingham E. Development and characterization of acellular porcine pulmonary valve scaffolds for tissue engineering. Tissue Eng Part A 2014;20:2963-74.

11) Boldt J, Lutter G, Pohanke J, Fischer G, Schoettler J, Cremer J, et al. Percutaneous tissue-engineered pulmonary valved stent implantation: comparison of bone marrow-derived CD133+-cells and cells obtained from carotid artery. Tissue Eng Part C Methods 2013;19:363-74.

12) Bond R, Rerkasem K, Naylor AR, Aburahma AF, Rothwell PM. Systematic review of randomized controlled trials of patch angioplasty versus primary closure and different types of patch materials during carotid endarterectomy. J Vasc Surg 2004;40:1126-35.

13) Inoue T, Croce K, Morooka T, Sakuma M, Node K, Simon DI. Vascular inflammation and repair: implications for re-endothelialization, restenosis, and stent thrombosis. JACC Cardiovasc Interv 2011;4:1057-66.

14) Bai H, Kuwahara G, Wang M, Brownson KE, Foster TR, Yamamoto K, et al. Pretreatment of pericardial patches with antibiotics does not alter patch healing in vivo. J Vasc Surg 2014;pii:S0741-5214(14)01862-X.

15) Strange G, Brizard C, Karl TR, Neethling L. An evaluation of Admedus’ tissue engineering process-treated (ADAPT) bovine pericardium patch (CardioCel) for the repair of cardiac and vascular defects. Expert Rev Med Devices 2015;12:135-41.

16) Gale NW, Holland SJ, Valenzuela DM, Flenniken A, Pan L, Ryan TE, et al. Eph receptors and ligands comprise two major specificity subclasses and are reciprocally compartmentalized during embryogenesis. Neuron 1996;17:9-19.

17) Swift MR, Weinstein BM. Arterial-venous specification during development. Circ Res 2009;104:576-88.

18) Kuijper S, Turner CJ, Adams RH. Regulation of angiogenesis by Eph-ephrin interactions. Trends Cardiovasc Med 2007;17:145-51.

19) Rockman CB, Halm EA, Wang JJ, Chassin MR, Tuhrim S, Formisano P, et al. Primary closure of the carotid artery is associated with poorer outcomes during carotid endarterectomy. J Vasc Surg 2005;42:870-7.

20) Hristov M, Weber C. Endothelial progenitor cells in vascular repair and remodeling. Pharmacol Res 2008;58:148-51.

21) Burt RK, Testori A, Oyama Y, Rodriguez HE, Yaung K, Villa M, et al. Autologous peripheral blood CD133+ cell implantation for limb salvage in patients with critical limb ischemia. Bone Marrow Transplant 2010;45:111-6.

22) Hristov M, Weber C. Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. J Cell Mol Med 2004;8:498-508.

23) Si TG. The experimental study of relation between sex hormone and restenosis after angioplasty. [Doctoral dissertation], Tianjin: Tianjin Medical University; 2003.

24) Lee JM, Choe W, Kim BK, Seo WW, Lim WH, Kang CK, et al. Comparison of endothelialization and neointimal formation with stents coated with antibodies against CD34 and vascular endothelial-cadherin. Biomaterials 2012;33:8917-27.

25) Ederle J, Dobson J, Featherstone RL, Bonati LH, van der Worp HB, de Borst GJ, et al. Carotid artery stenting compared with endarterectomy in patients with symptomatic carotid stenosis (International Carotid Stenting Study): an interim analysis of a randomised controlled trial. Lancet 2010;375:985-97.