Heterotopic Xenotransplation of Newborn Piglet Aortic Fragments After Hypothermal Incubation in Preservation Solutions

Nowadays the porcine vessels and heart valves have been widely applied in transplantology. Due to a high level of Gal-α-1,3-Gal (α-Gal epitope) expression, the porcine bioprostheses are specially treated prior to transplantation either to remove this epitope or to deactivate it. Our previous findings showed that certain media, used for hypothermic storage (HS), reduced the α-Gal epitope expression in aortic tissue in vitro. Here, we studied the HS impact in the histidinetryptophan-ketoglutarate (HTK), Euro-Collins and Dulbecco’s Modified Eagle Medium (DMEM) solutions on aortic graft integrity in vivo. The analysis of aortic wall histological structure and the evaluation of the immunoproteasome subunits LMP2 and LMP7 expression level demonstrated the aortic fragments, subjected to HS in Euro-Collins solution to day 7 after xenotransplantation to cause a less pronounced immune response as compared to the HTKand DMEM-incubated xenografts. This may be partially due to the Euro-Collins medium composition. Our findings may be of interest for elaborating the novel methods for donor material pre-transplantation preparation.

During evolution, the gene responsible for the α-1,3-galactosyltransferase (1,3-GT) enzyme expression has been inactivated in human and some monkey species, therefore there is no α-Gal epitope contained in human cell membrane glycoproteins [15].However, this enzyme is expressed in cells of bacteria, which colonize the human intestine.As a result, there is a high titer of antibodies to the α-Gal epitope in human blood [14].
The α-Gal epitope was established to be recognized during xenotransplantation not only by means of antibodies [19], but via antigen presenting either by dendritic cells or macrophages (antigen presenting cells (APCs)) to CD8 + T-lymphocytes as well [10].According to current concepts, the immunoproteasomes are involved into antigen processing for cross-presentation [7,18,31,32].Consequently, the expression of immunoproteasome subunits in the transplantation site may be one of the signs of recognition and presentation of foreign antigens.
It is known that the substances within either preservation or perfusion media may aff ect the cell membrane and modify its surface [17,26].The current technologies for processing the bioprostheses, suitable for transplantation, include the obligatory steps of either α-Gal epitope removal or deactivation [5,13].
Several authors found an enhanced survival in grafts after prolonged hypothermic perfusion with cardioplegic solution, incubation with a cryoprotectant or cryopreservation [3,6,38].M. Keller et al. indicated a decrease in α-Gal epitope expression in porcine aortic endothelial cells after incubation in HTK solution [21].The in vitro experiments demonstrated a decrease in α-Gal expression level in the newborn piglet aortic tissue after 24-hour incubation in HTK and Euro-Collins solutions at 4°C [2].However, the way how a decrease in α-Gal epitope expression on aortic cell membranes aff ects the survival of tissue after in vivo xenotransplantation, has not yet been fully elucidated.
The research was aimed to study the eff ect of hypothermic incubation of newborn piglet aorta in preservation media of diff erent composition on the integrity of its histological structure and the проблеми кріобіології і кріомедицини problems of cryobiology and cryomedicine том/volume 29, №/issue 1, 2019 нах клеток аорты на выживаемость ткани после ксенотрансплантации in vivo.

Materials and methods
The experiments were carried out in accordance with the Law of Ukraine On the Protection of Animals Against Cruelty (№ 3447-IV of February 21, 2006), in compliance with the requirements of the Bioethics Committee of the Institute for Problems of Cryobiology and Cryomedicine of the NAS of Ukraine, agreed to the statements of European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientifi c Purposes (Strasburg, 1986).
The newborn piglet aortic fragments of 0.5-0.7 cm long were cut lengthwise, washed in cold sterile physiological saline and then placed for 20 hrs at 4°C into either HTK (Dr.F. Kohler Chemie GmbH, Germany) or Euro-Collins (Chemech Laboratories Ltd, India) solutions.
The aortic fragments, incubated in DMEM (PAA, Austria) for 20 hrs under the same conditions, as well as the aortic tissue, treated with α-galactosidase enzyme ('α-Galactosidase from green coff ee beans', Sigma, USA) were the reference specimens.This enzyme is known to eliminate the α-Gal epitope from the porcine cell surface [29].
The treatment with enzyme was as follows.An aortic fragment (0.5×0.5 cm) was incubated in α-galactosidase enzyme solution (5 U/ml of enzyme, 100 mM HEPES, pH 5.0) for 20 hrs at 4°C, then washed three times with sterile saline for 5 min.
An ectopic xenotransplantation was carried out to 6-8-month-old outbred white male rats under the renal capsule according to the method of Robertson et al. [30].The aortic fragments were washed of the preservation and enzymatic media by sterile physiological saline, cut with scalpel into pieces of approximately 0.1 × 0.1 cm and transplanted under the left renal capsule.During surgery the rats were anesthetized with Zoletil 100 (Virbac, France) and Sedazin (Biowet, Poland) combination.No immunosuppressive drugs were used in preand post-transplantation periods.
The animals were divided into four groups: the group 1 comprised the animals with xenotransplantation of fragments after HS in HTK (n = 5); the group 2 consisted of the animals with xenotransplanted fragments after HS in Euro-Collins (n = 5); the fragments post HS in DMEM made the group 3 (n = 5); those after α-galactosidase treatment (n = 4) represented the group 4.
For immunofl uorescent labeling of the immunoproteasome subunits LMP2 and LMP7 the specimens were permeabilized in 0.1 M PBS (pH 7.4) containing 0.25% Triton X-100 and 0.1% Tween 20 (both Sigma) for 20 min.To block the non-specifi c labeling, the sections were kept for 60 min at room temperature in the solution, containing 2% bovine serum albumin (Sigma), 0.2% Triton X-100, and 0.3 M glycine (Reanal, Hungary).Then the specimens were incubated at 1:500 dilution either with primary rabbit antibodies to LMP7 subunit (Enzo Life Science, USA) or mouse antibodies to LMP2 subunit (Enzo Life Science) for 15 hrs at 4°C.After a threefold wash-out of primary antibodies, the specimens were incubated for 30 min at room temperature with the secondary antibodies: antirabbit Alexa 488-conjugated (Abcam, UK) at 1:700 dilution or anti-mouse Alexa 546-conjugated (Abcam) at 1:600 dilution, respectively.For macrophage labeling, a direct labeling protocol with PE-conjugated antibodies (Anti-Rat Macrophage Marker, eBioscience, USA) at 1:60 dilution, was used.
The data were statistically processed using the Excel (Microsoft, USA) and Statistica 7.0 (StatSoft, USA) software.The data were presented as the mean -standard deviation.Diff erences between the samples were assessed with the univariate analysis of variance, the diff erences were considered signifi cant at p < 0.05.

Results and discussion
The donor tissue transplantation under the renal capsule of recipient is widely used in experimental transplantology, since the subcapsular space is not an immunologically privileged site, and it allows grafting a suffi cient amount of donor tissue and ensures a good vascularization of the graft [30].
Here, we used a subcapsular transplantation to assess the survival of aortic tissue, its morphological features, and the intensity of immunological reactions, developed in response to xenograft.
The analysis of the kidneys with xenografts revealed the preserved histological structure of aortic tissue to day 7 post transplantation (Fig. 1).In the specimens of all the experimental groups, we could see the tunica media of the aorta, represented by parallel strands of elastic elements.The renal capsule fi bers over the grafts were often disarranged, with single small infi ltrates.The rest of the kidney was of normal appearance, the tubular epithelium was preserved.
Specifi c structural changes were revealed in xenografts depending on pre-transplant treatment of donor tissue.In the group 1 xenografts (fragment incubation in HTK solution), the aortic elastic fi bers were convoluted and had small gaps in-between.Lymphohistiocytic infi ltration was represented by small single cell clusters in graft-adjacent parenchyma.
In the group 4 xenografts (α-galactosidase-processed fragments), the aortic elastic fi bers were convoluted with the minor gaps in-between.Lymphohistiocytic infi ltration was slightly pronounced.
The Table 1 demonstrates the results of cell quantitative analysis in the infi ltration foci in the xenograft and at the boundary with the kidney parenchyma.It is seen, that the number of infi ltrating cells is signifi cantly lower in the xenograft sections of groups 2 and 4 than in groups 1 and 3.In general, the qualitative analysis of the aortic xenograft state and quantitative assessment of focal infi ltration cells testify to a decrease in immune response to aortic tissue, subjected to enzymatic treatment with α-galactosidase and HS in Euro-Collins solution.
The labeling with antibodies to the immunoproteasome subunit LMP7 showed a signifi cant number of positively stained cells, observed in the aortic xenografts, subjected to HS in HTK (group 1) and DMEM (group 3) (Fig. 2B, D; Table 2).No pronounced expression of LMP7 was found both at the boundary with xenograft and at the rest kidney parenchyma.
In the Euro-Collins-treated aortic specimens (group 2), no LMP7 labeling in xenograft thickness was observed, but it was present in renal parenchyma (Table 2).
Labeling with antibodies to the immunoproteasome subunit LMP2 revealed the similar features in all the groups, i. e. a slight expression in xenografts and a high in renal parenchyma (Fig. 3A, B; Table 2).It is characteristic, that in renal parenchyma, a positive labeling was observed mostly in tubular epithelium, and not in Malpighian bodies.
Here, we selected the solutions with crucially diff erent composition such as: -the Euro-Collins, which simulated intracellular electrolyte composition due to a high content of potassium ions and contained the glucose, providing an osmotic barrier; -HTK, containing low concentrated potassium and sodium, which comprised mannitol, acting as an osmotic barrier, as well as the amino acids, stabilizing cell membranes and being the substrates for anaerobic metabolism; -DMEM, simulating the composition of extracellular medium (low potassium and high sodium levels), used in cell culture technologies.
Initially, when developing the composition of preservation media, the researchers did not set the task to modify the antigenic profi le of tissues/ organs to be transplanted.Nevertheless, during their use the facts appeared, which testifi ed to the impact of these media on immunogenicity of the transplanted organs.P. Brenner et al. [6] noted that prolonged hypothermic perfusion of porcine heart with a preservation solution entailed a decrease in the immune response to xenograft.More detailed experiments performed by M. Keller et al. [21] in porcine aortic endothelial cell culture showed a decrease in α-Gal epitope expression on cell surface by 32% after incubation in HTK.In our previous work, a comparative immunofl uorescent labeling of the α-Gal epitope after HS of newborn piglet aortic fragments in HTK and Euro-Collins (24 hrs) showed a decrease in this index as compared to the fresh specimens [2].
It is of interest that in the group 2 specimens, we obtained the results similar to group 4, that testifi ed to a decrease in the aortic tissue immunogenicity after HS in Euro-Collins solution.Much more pronounced signs of rejection, manifested in a partial destruction of xenograft and an increased number of infi ltrating cells, were observed in specimens of the remaining groups.
M. Keller et al. [21] suggested the following possible molecular mechanisms for reducing the presence of α-Gal epitope on cell membranes: -changes in chemical structure of the epitope or associated protein, which made it impossible to recognize them with specifi c antibodies; -the epitope elimination from cell membrane due to the breaking of intermolecular bonds; -the epitope masking with high-molecular substances, being the part of preservation media.
Moreover, Keller M. observed a signifi cant modifying eff ect of the University of Wisconsin (UW) solution on the α-Gal epitope, due to the masking of the epitope by the high-molecular compounds, i. e. starch, raffi nose and lactobionate [21].In our study, a modifying eff ect was observed when using the Euro-Collins solution, which did not contain such substances.Nevertheless, both solutions, Euro-Collins and UW, contain a high concentration of potassium ions.Possibly, this factor contributes to the α-Gal epitope modifi cation.Thus, the additional experiments are needed to clarify this mechanism.
To date, the role of proteasomes has been proven in the immune response development as the specialized multisubunit protein complexes, performing an intracellular proteolysis.The genes, encoding the immune subunits LMP2 and LMP7 are located in the locus of the major histocompatibility complex (MHC) class II [27].When these subunits are built into a proteasome, its proteolytic activity changes and the production of antigenic epitopes increases.The captured by APCs antigen undergoes the proteolysis by immunoproteasomes in the cytoplasm, then transferred into endoplasmic reticulum by means of TAP complex (Transporter associated with Antigen Processing), loaded into the MHC class I and transferred onto the membrane surface for presentation.The immunoproteasomes are involved in antigen cross-presentation through MHC class I, resulting in immune response activation by cytotoxic CD8 + T-lymphocytes [22,32].тикулум с помощью TAP-комплекса (Transporter associated with Antigen Processing), загружается в комплекс ГКГ класса I и переносится на поверхность мембраны для презентации.Иммунопротеасомы участвуют в кросс-презентации антигенов посредством ГКГ класса I, приводя к активации иммунного ответа цитотоксическими CD8 + Т-лимфоцитами [22,32].
It was previously established the fact, that the grafts, subjected to either prolonged cold ischemia [9] or ischemia-reperfusion [24], even in the fi rst fi ve days after transplantation were infi ltrated by neutrophils and macrophages.The primary triggers for such an activation are the molecular signals of damage, the DAMPs (damage-associated molecular patterns) [23], which inevitably appear in grafts due to cell death during their isolation and storage.In addition, the surgery is associated with activation of the macrophages and their recruitment into the transplantation site due to a local increase in chemotactic factors MIP-1, MCP-1 and RANTES, released from damaged vascular cells and parenchyma [37].A locally increasing concentration of IFN-γ and other pro-infl ammatory cytokines, secreted by macrophages at the transplantation site entails the expression activation of the immunoproteasome subunits [25].Herewith, under the impact of cytokine stimulation, the immune subunits may appear not only in immunocompetent cells, but in parenchymal ones as well [11].
For example, H. Ostrowska et al. [28] proved the induction of immunoproteasome synthesis in renal cells after prolonged ischemia and under infl ammation.In our investigations, we observed an increased expression of immune subunit LMP7 in renal parenchyma after HS in Euro-Collins solution, but not in other media.This fact may result from a specifi c impact of certain components of Euro-Collins solution, that requires an additional study.
Obviously, during xenotransplantation of aorta, subjected to HS, a complex of activation signals is initiated, including the response to surgery, the release of DAMPs from the own tissues and from the transplanted aorta, the secretion of pro-infl am- зано со специфическим влиянием некоторых компонентов, входящих в состав «Евро-Коллинз», и требует отдельных исследований.
3. Ткань аорты новорожденных поросят, подвергнутая инкубации в растворе «Евро-Коллинз» при 4℃, вызывает менее выраженный иммунный ответ по сравнению с ксенографтами, инкубированными в растворах НТК и DMEM.and DMEM solutions, as the formation of foci of lymphohistiocytic infi ltration and an increased expression of immunoproteasome subunits at the transplantation site.At the same time, a decrease in the number of infi ltrating cells and the lack of expression of subunits LMP2 and LMP7 in xenografts after HS in Euro-Collins solution indirectly indicates not only quite a good preservation of the tissue, but a decreased expression of xenoantigens in it as well.
Thus, the presented in vivo study confi rms our previous in vitro fi ndings, showing that a decrease in the α-Gal epitope expression level as a result of HS of piglet aortic fragments in Euro-Collins solution leads to a less pronounced immune response of recipient during xenotransplantation.Nevertheless, the molecular mechanisms for reducing the xenoantigen expression have not been fully studied yet and further research is needed.Conclusions 1.To day 7 after heterotopic xenotransplantation under the renal capsule, the newborn piglet aortic tissue preserved its histological structure.However, in xenografts we observed the foci of lymphohistiocytic infi ltration, the intensity of which depended on incubation medium.
2. Xenotransplantation of the newborn piglet aortic fragments under renal capsule to rats results in an increase in the expression of the immunoproteasome subunits LMP2 and LMP7 in the xenograft, associated with the infi ltration foci formation.
3. The newborn piglet aortic tissue, subjected to incubation in Euro-Collins solution at 4°C causes a less pronounced immune response as compared to the xenografts, incubated in HTC and DMEM.

Table 2 .
Semi-quantitative indices of labeling of cells, expressing LMP7, LMP2, Anti-Rat Macrophage Marker per 1,000 μm 2 of the section area in aortic xenograft (fi eld 1), renal parenchyma at the boundary with xenograft (fi eld 2), as well as in rest kidney parenchyma (fi eld 3)