Plasmon-Activated Water Decreases Vasculopathy in Orthotopic Allograft Transplantation Rats

Patients undergoing orthotopic allograft transplantation (OAT) will certainly suffer from vasculopathy. Although there are many immunosuppressive and immunomodulatory agents that are administered to patients, chronic rejection- induced vasculopathy cannot be entirely managed. Moreover, the implanted graft might become dysfunctional. In the past, we have used deionized reverse osmosis water (ROW) to stream via gold nanoparticles (AuNPs) at room temperature under powerful illumination, in order to prepare plasmon-activated water (PAW) with fewer hydrogen bonds. Compared to ROW, stable PAW can successfully remove free hydroxyl and 2,2-diphenyl-1-picrylhydrazyl radicals, and eciently reduce lipopolysaccharide (LPS)-induced monocytes to release nitric oxide. Moreover, PAW can considerably induce the expression of the antioxidant gene Nrf2 in human gingival broblasts. Moreover, it might lower amyloid burden in mice with Alzheimer's disease. Furthermore, PAW decreased metastasis in mice grafted with Lewis lung carcinoma cells and boosted the overall survival in combination with cisplatin. Because of this possibility that PAW could prevent systemic disease, we aimed to evaluate the inuence of PAW on OAT-induced vasculopathy. Here, we demonstrated that daily intake of PAW lowered the progression of vasculopathy in OAT-recipient ACI/NKyo rats by inhibiting collagen accumulation, proliferation of smooth muscle cells and broblasts, and T lymphocyte inltration in the vessel wall. Moreover, the results showed reduced T and B lymphocytes, plasma cells, and macrophage activation in the spleen of the OAT-recipient ACI/NKyo rats that were administered PAW. Finally, in contrast to the control group, the OAT-recipient ACI/NKyo rats that were administered PAW exhibited higher mobilization and levels of circulating endothelial progenitor cells associated with vessel repair. Therefore, this study highlights the therapeutic roles of PAW in vasculopathy. therapeutic effects than current treatment modalities. Overall, this study indicated that daily consumption of PAW might decrease the progress of vasculopathy in OAT-recipient ACI/NKyo rats by promoting the inhibition collagen accumulation, SMC and broblast inltration, and Tc and Th cell responses in the vessel walls. Furthermore, the data showed reduced activation of T lymphocytes, B lymphocytes, plasma cells, and macrophages in the spleen of PAW administered OAT-recipient ACI/NKyo rats compared to ROW administered OAT-recipient ACI/NKyo rats. Finally, we observed higher mobilization and levels of circulating EPCs in PAW-administered OAT-recipient ACI/NKyo rats compared to control group. Therefore, this study highlights the potential therapeutic roles of PAW in OAT-induced vasculopathy.


Introduction
Orthotopic allograft transplantation (OAT) is the main treatment method for end-stage organ failure.
Since the currently available clinical strategies can effectively control acute rejection, short-term survival rate of patients after organ transplantation has greatly improved. However, presently, the main factor affecting the long-term survival of patients undergoing organ transplantation is chronic rejection. Chronic rejection is a process in which the alloimmune system attacks the donor graft, and causes continuous and diffused damage of the vessels, resulting in vasculopathy 1 . The targets of this alloimmune system attack include the epithelium and endothelium of the implanted graft. Arteries and microvessels, which cause the original normal parenchyma to be replaced by brotic scar, exhibit the hyperplasia of vascular intimal brosis that causes diffuse occlusion and narrowing of the vessels implanted in the graft 2 . Eventually, progressive arterial brosis and constriction will lead to organ ischemia, with the lesions leading to organ failure in patients after transplantation 3 . Therefore, OAT-induced vasculopathy is an important problem after organ transplantation that needs to be resolved.
The occurrence of chronic rejection after OAT is indeed related to the production of antibodies against donor-speci c human leukocyte antigen (HLA) by the recipient 4 . Since OAT-induced vasculopathy only occurs in the vessels of the donor grafts that are transplanted, but not in the native vessels of the recipients, alloimmunity is currently considered to be the main cause of OAT-induced vasculopathy 2 .
Donor antigen-presenting cells present in the donor organ, such the MHC molecules on the surface of dendritic cells, will be recognized by the recipient's T cells, which, in turn, results in the activation of the cellular immune response. In addition, the donor antigen fragments presented on the surface of the recipient's antigen-presenting cells will be recognized by the recipient's T cells 3 . This process is the main initiator of the immune response responsible for chronic rejection 5 . The activated T cells will release interleukin-2 (IL-2) and interferon-γ (IFN-γ). These cytokines will continue to activate the T cells, B cells, and macrophages, and additionally, the graft vascular endothelial cells will also be activated and will begin to express several vascular cell adhesion molecules. This process causes an increased number of recipient immune cells to be chemoattracted to the vascular endothelium of the donor graft, thereby amplifying the immune response 6 . The smooth muscle cells are also activated simultaneously, resulting in their proliferation 7 , the secretion and accumulation of extracellular matrix proteins, and concomitantly, the activated B cells produce anti-HLA antibodies, which combine with macrophages to jointly regulate the vascular damage of the implanted graft. Therefore, the damage after OAT, resulting from both T cellassociated cellular immune responses and B cell-associated humoral immune responses 5 , eventually leads to vascular intimal hyperplasia, brosis 8 , ischemia, and functional failure of theimplanted graft 9 .
Plasmon-activated water (PAW) is produced by allowing deionized reverse osmosis water (ROW) to ow through gold nanoparticles (AuNPs) under resonant illumination, which excites the hot electron transfer provided by AuNPs and destroys the hydrogen bonds between H 2 O 10, 11 . Compared with ROW, PAW has higher physical and chemical activity, including greater solubility for alkali metal-chloride salts, increased ionic conductivity of NaCl, higher extraction e ciency of biological antioxidants, such as polyphenols, 2,3,5,4'-tetrahydroxystilbene-2-O-beta-d-glucoside (THSG) and polygonum multi orum 12 . Intracellular ROS production and ROS-activated signaling are closely associated with the development of cancers, autoimmune diseases, and systemic in ammatory diseases. In recent years, scientists have found that PAW might reduce ROS-induced cellular and tissue damage mediated by various mechanisms 13 . In addition, PAW can inhibit the lipopolysaccharide (LPS)-induced production of nitric oxide (NO) by monocytic cells, thereby inhibiting oxidative stress during acute in ammatory responses 10 . It might also activate the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2 related factor 2 (Nrf2)/antioxidant response element (ARE) pathway and increase the antioxidant capacity of human gingival broblasts. Additionally, the administration of PAW may increase the survival rate of non-small cell lung cancer (NSCLC)-bearing mice that are administered cisplatin and reduce the metastasis of NSCLCs 14 . Furthermore, it might also reduce the myeloid burden in neurons and improve memory as well as decrease the severity of Alzheimer's disease in APPswe/PSEN1dE9 transgenic mice 15 .
Because PAW is safe, easily produced, and inexpensive, it should be a publicly accessible and an fesible option. Based on our previous ndings regarding the effectiveness of PAW in the treatment of in ammation-related diseases, we planned to further evaluate the effect of PAW on the occurrence of OAT-induced vasculopathy. In this study, we transplanted the thoracic aorta of PVG/Seac rats into the abdominal aorta of ACI/NKyo rats and compared the severity of vasculopathy after 30-150 days of OAT in animals that were administered ROW and PAW. We also compared the chronic rejection situation of animals in the two groups by immunohistochemical analysis of the spleen and explored the underlying mechanisms of OAT-induced vasculopathy. We expect that this study will enhance the applicability of PAW in the prevention and treatment of in ammatory diseases in the future.

Results
Administration of PAW did not affect the biochemical characteristics in OAT ACI/NKyo rats Biochemical analyses were performed to evaluate the effects of PAW on OAT ACI/NKyo rats. As shown in Table 1, body weight change, BUN, creatinine, ALT, and AST did not differ among the groups during the experimental period. PAW decreases vasculopathy and collagen accumulation in OAT ACI/NKyo rats Representative photographs of vasculopathy in the thoracic aortas (from donor PVG/Seac rats) stained with H&E were shown in Fig. 1A, and the quanti cations of intima/vessel wall area ratio are shown in Fig. 1B. No vasculopathy signs were observed in the aortas of normal PVG/Seac rats. Severe damage to vascular integrity, blurred elastin laminae, and accumulation of calci ed plaques were markedly increased in the OAT group compared to normal vessels in PVG/Seac rats (Fig. 1A). Additionally, the formation of an incomplete vascular structure correlated with time (from day 30 to day 90) in the OAT group. A signi cantly smaller area of aortic vasculopathy and greater vascular integrity were observed in the OAT plus PAW group compared to that of the OAT plus ROW group on day 90. Interestingly, the vessel wall remained intact in addition to slight neointimal formation at day150 after OAT, and there was no calci ed plaque formation.
Vasculopathy has been implicated in the development of collagen accumulation 16 . Therefore, picrosirius red staining was performed for visualizing collagen. As shown in Fig. 1C, normal thoracic aortas from PVG/Seac rats presented intact, thick collagen bers (weak orange to red) and an equal distribution of ne collagen bers (yellow to green) in the vessel walls. In contrast, the vessels in the OAT with ROW group at day 90 appeared to exhibit wall thickening in the inner layer, with some of the ne collagen bers being piled up with a small amount of messy, thick collagen bers. Moreover, the thick collagen bers in the original vessel walls became inconsistent in thickness. However, in the OAT with PAW group, the disordered distribution and accumulation of ne and thick collagen bers indeed improved signi cantly compared to that of the OAT with ROW group at day 90. The vessel wall showed reduced collagen accumulation which lasted up to day 120 and day 150 in the OAT plus PAW group.
Reduced proliferation and accumulation of immune cells was observed in the vessel wall of PAW administered OAT ACI/NKyo rats Proliferation of SMCs and broblasts plays a critical role in the process of chronic allograft vasculopathy. Therefore, immunohistochemical staining was performed using antibodies against αSMA and S1000A4 on transplanted aorta sections ( Fig Administration of PAW results in lower plasma concentration of in ammatory factors and cytokines in OAT ACI/NKyo rats As shown in Table 2, in ammation-related cytokines and markers were produced in the plasma. LDH is an enzyme that is released into the plasma following tissue injury, and might be strongly associated with antibody-mediated rejection 17 . Elevated plasma levels of LDH were observed in OATACI/NKyo rats administered with ROW; higher LDH levels (1888.78 ± 323.0 IU/L) compared to the baseline (854.6 ± 152.5 IU/L) were observed in the ROW group; the level of LDH was signi cantly higher than that of the non-OAT group (895.9 ± 125.6 IU/L) at day 90. However, the elevated LDH level was decreased in the OAT plus PAW group (968.8 ± 116.4 IU/L) at day 90.CRP is an indicator of in ammation and tissue damage 18 and HMGB1 is involved in the chronic rejection of cardiac allograft vasculopathy 19 . As shown in Table 2  OAT, orthotopic aortic transplantation; LDH, lactic dehydrogenase; CRP, C-reactive protein; HMGB1, high mobility group box 1 protein; INF-γ, interferon γ; TGF-β1, transforming growth factor-beta 1; SDF-1α, stromal cell-derived factor 1α; IL-2, interleukin 2; Values are represented as mean ± SD. a p < 0.05 compared with baseline of the same group; b p < 0.05 compared with control ACI/NKyo (non-OAT) group at the same time point; c p < 0.05 compared with OAT + ROW (PVG/Seac to ACI/NKyo) group at the same time point. Statistical evaluations were performed using one-way ANOVA.
Administration of PAW results in reduced cell-mediated and humoral immune responses in OAT ACI/NKyo rats Humoral and cell-mediated immunity are associated with the process of OAT-induced vasculopathy.
Therefore, the spleens of experimental animals were analyzed by IHC to identify the severity of chronic rejection. CD11b positive macrophage, which is also an antigen-presenting cell, activates the adaptive immune system. 23 As shown in Fig. 3A, macrophages were barely observed in the germinal center (GC) and periarterial lymphatic sheath (PALS) of the spleen from naïve ACI/NKyo rat. In contrast, an increased number of macrophages appeared in the GC and PALS in the ROW administered group after OAT for 90 days. However, after 90 and 150 days of OAT, PAW administration signi cantly reduced the accumulation of macrophages in the GC and PALS. There are two major subtypes of T lymphocytes, helper T (Th) cells (CD4 + ) and killer T (Tc) cells (CD8 + ) involved in cell-mediated immunity. As shown in Fig. 3B, the GC and PALS of spleen of naïve ACI/NKyo rats did not accumulate CD4 positive Th cells. In addition, rats in the OAT plus ROW group presented increased accumulation of CD4 positive Th cells in GC and PALS of spleen, which decreased upon PAW administration. Similar to CD4 positive Th cells, CD8 positive Tc cells also in ltrated into the PALS of spleen in OAT plus ROW ACI/NKyo rat, which was reversed upon PAW administration. Additionally, B cells (CD20 + ) mediate humoral immune responses and differentiates into plasma cells (CD138 + ) to produce antibodies. As shown in Fig. 3C, CD20 positive B cells predominantly cluster in the germinal center and mantle zone, and simultaneously, reduced number of plasma cells were observed in the germinal center, mantle zone, and venous sinuses of the spleen of naïve ACI/NKyo rat. ACI/NKyo rat with OAT plus ROW administration presented a large number of CD20 positive B cells accumulated in the germinal center and mantle zone, and activated plasma cells were observed at venous sinuses. In ACI/NKyo rat with OAT plus PAW administration, longer duration of PAW administration resulted in decreased accumulation of CD20 positive B cells in the germinal center and mantle zone. However, plasma cells were not observed in the venous sinuses, germinal center, and mantle zone. Based on these results, we predict that administration of PAW might maintain low levels of cellmediated and humoral immune responses in ACI/NKyo rats.

Administration of PAW promotes increased mobilization of circulating EPCs and differentiation of early EPCs than administration of ROW in ACI/NKyo rats
EPCs play an important role in repairing damaged vessels during the process of OAT 24 . As shown in Table 2, administering rats with PAW after OAT resulted in increased SDF-1α and decreased TGF-1β as well as INF-γ production in ACI/NKyo rats. SDF-1α may mobilize circulating EPCs from the bone marrow 25 . TGF-1β is a guardian of T cell function 26 27 . Therefore, following OAT surgery, the population of endogenous EPCs (de ned as CD133 + /CD34 + /VEGF + cells) and SMPCs (de ned as CD133 + /αSMA + / CD34 − cells) were quanti ed by ow cytometry, to compare the difference in levels of these cells between ROW and PAW administered ACI/NKyo rats. The results showed a signi cant increase in EPCs after 30 days of OAT in ROW administered ACI/NKyo rats compared to that of non-OAT ACI/NKyo rats, and this increase was maintained until day 90 after OAT (Fig. 4A). At day 30, OAT ACI/NKyo rats from the PAW administered group exhibited a signi cantly increased number of mobilized EPCs in the peripheral blood compared to that of OAT plus ROW group. However, the number of circulating EPCs was not associated with the time point after OAT in both ROW and PAW administered ACI/NKyo rats. Moreover, SMPCs initiated transplant arteriosclerosis. However, as shown by ow cytometry, the number of circulating SMPCs was not related to water administration, regardless of whether the animals received OAT (Fig. 4B).
The capacity of MNCs to form EPC colonies may represent the differentiation ability of circulating EPCs. As shown in Fig. 4C, the MNCs from ACI/NKyo rats seeded on bronectin-coated wells on day 7 appeared to form early EPC colonies. MNCs from OAT ACI/NKyo rats administered with ROW and PAW also formed EPC colonies at 7 days of culture. The colonies of EPCs appearing on day 7 of culture presented as a central core of round cells with elongated sprouting cells at the periphery. However, rats receiving PAW exhibited more e cient differentiation of MNCs. After 21 days of continuous culture, EPCs had cobblestone-like morphology similar to that of mature endothelial cells and were con uent in the PAW administered group. Conversely, the cells were sparse in the ROW administered group (Fig. 4C). These results indicated that PAW administration promotes increased mobilization and differentiation of early circulating EPCs than administration of ROW in OAT-recipient ACI/NKyo rats.

Discussion
Water plays a vital role in all living entities and features as a solvent. In the human body, water functions as a medium for nutrient transfer and stabilizes body temperature levels. Additionally, water takes part in certain biochemical reactions, such as hydrolysis 28 , glycogen disintegration, and adenosine triphosphate disintegration 29 . Water, a widely utilized solvent, is typically considered inert in chain reactions. Nevertheless, water with its characteristic property of donor bridge acceptor for proton as well as electron transfer has been considered as an appealing energetic reactant [30][31][32] . Surface plasmon resonance causes excitation of AuNPs to decay into energized hot electrons. Hot electron transfer can promote numerous chemical reactions 33 , including dissociation of hydrogen 34 , activation of oxygen 35 , as well as electrocatalytic hydrogen evolution 36 . Our group proposed an innovative method to create PAW, which is prepared by treating ROW with resonantly excited AuNPs 10,37,38 . Recently, we have demonstrated that PAW is innovatively applicable in various elds 39 . PAW with its unique properties of enhancing solute diffusion as well as exhibiting anti-in ammatory properties considerably shortens the time required to remove uremic contaminants during hemodialysis therapy 11,40 . Additionally, PAW decreased bronectin expression and renal brosis in mice with chronic kidney disease 40 , and reduced liver damage in chronic sleep-deprived rats 41 . Compared to mice administered with ROW, PAW administration may improve the survival time and cisplatin effect in Lewis lung carcinoma cells-bearing mice, and may be associated with inducing Nrf2 gene expression 14 . A previous study suggests that PAW has potential therapeutic effects against Parkinson's disease, periodontal disease, and Alzheimer's disease 15 .
After organ transplantation, once the immune and in ammatory responses are activated by alloimmune-, autoimmune-, or non-immune factors, the brotic changes that occur in the arteries of the graft results in OAT-induced vasculopathy, which subsequently causes ischemia 42 . Endothelium muscularization and intimal hyperplasia can be observed in most transplanted organs 43 . However, endo-MT refers to the process of differentiation of endothelial cells into interstitial cells (such as smooth muscle cells and broblasts) 44 , which has been considered to be associated with the occurrence of OAT-induced vasculopathy [44][45][46] . A previous report has demonstrated that smooth muscle cell (SMCs) proliferation, mobilization and differentiation of circulating EPCs, and endo-MT co-regulate the process of OAT-induced vasculopathy. The arteries of OAT-induced vasculopathy indicate the presence of SMCs which are mostly derived from the donor 47 and are activated and proliferate following in ammatory responses. These cells exhibit chemotaxis moving from the media to the intima of the vessel 9 . In addition, donor-derived SMCs at the lesion originate from donor-derived ECs formed via endo-MT 48 . In recipients, bone marrowderived CD133 + /CD34 + /VEGF-R1 + EPCs and circulating CD31 + /CD146 + /vWF + /NOS + EPCs have the characteristics of endothelial regenerative properties [49][50][51] . However, recent studies have found that recipient's EPCs are, instead, involved in the pathological process of vasculopathy 5,50,52 due to the fact that circulating EPCs will adhere to the arterial endothelium and begin to proliferate, and induce persistent allogeneic immune responses 52 . In addition, EPCs will uncontrollably accumulate and induce massive proliferation of SMCs, and the proliferating SMCs will secrete large amounts of extracellular matrix proteins to cause intimal hyperplasia 50 . Therefore, scientists speculate that despite the recipient exhibiting vigorous mobilization of EPCs, the persistent and uncontrolled allogeneic immune response may activate endo-MT and stimulate EPCs to differentiate into SMCs, which nally causes vasculopathy [52][53][54] . The TGF-β signaling pathway is closely associated with the induction of brosis 44,55 . In our study, we found that OAT increases the plasma level of SDF-1α (Table 2) as well as the number of circulating EPCs in ACI/NKyo rats. However, PAW administered ACI/NKyo rats had higher concentration of plasma SDF-1α and increased number of circulating EPCs compared to ROW administered rats (Fig. 4A).
Although TGF-β1 is the major factor that induces endo-MT, the plasma TGF-β1 concentration in OAT-ACI/NKyo rats fed with PAW was lower than that of the ROW fed group. Moreover, the decrease in circulating SMPCs in the PAW group was not statistically signi cant. We speculate that PAW may induce increased mobilization of EPCs from the bone marrow by increasing plasma SDF-1α concentration. On the other hand, PAW also reduces TGF-β1, including TGF-β1-mediated endo-MT which may be the possible mechanism of reducing OAT-induced vasculopathy. Endo-MT is regulated by complex molecular mechanisms. TGF-β1 and TGF-β2 promote endo-MT 54,56 . When endo-MT is induced by TGF-β, bone morphogenetic protein (BMP) and activin receptor-like kinase, regulate gene expression through phosphorylation of SMAD [60]. The phosphorylated SMAD enters the nucleus and interacts with key transcription factors that regulate endo-MT such as SNAI1, SNAI2, ZEB1, ZEB2, KLF4, TCF3, and TWIST which ultimately leads to chromatin rearrangement and endo-MT-related gene expression 57 . In addition to TGF-β mainly activating SMAD to initiate the process of endo-MT, it may activate other complementary pathways including mitogen-activated protein kinase pathway, phosphoinositide 3-kinase pathway, and inhibit the microRNA pathway that are all related to endo-MT. Therefore, we further intend to analyze the molecular mechanisms involved in PAW-induced inhibition of endo-MT.
In this study, we also analyzed the effect of PAW on the plasma levels of IFN-γ, HMGB1, and IL-2 in OATrecipient rats. It is well known that IFN-γ expression is associated with activation, differentiation, and recruitment of T-cells via direct or indirect signaling pathways 58,59 , which mediate OAT-induced vasculopathy. In 2016, Van Loosdregt and colleagues demonstrated that patients with transplanted heart vasculopathy had higher levels of IFN-γ, IFN-γ-inducible chemokines (fractalkine/CX3CL1, RANTES, ITAC, and IP-10), and IFN-γ-secreting Th1 cell markers (CXCR3, CCR5, and CX3CR1) in coronary arteries compared to control individuals 60 . Additionally, the nuclei of necrotic cells may release HMGB1 61 ; the mature antigen-presenting cells and macrophages are stimulated by IFN-γ or TNF-α to secrete HMGB1 62,63 . In macrophages, the secretion of HMGB1 induces hyperacetylation of the protein 64 and increases the ability to participate in adaptive alloimmune rejection. HMGB1 may activate the differentiation of Th1 cells via receptor for advanced glycation end-products (RAGE) 62,65 . Stimulated endothelial cells in the vessel wall also produce HMGB1, and subsequently activate more endothelial cells and amplify the in ammatory responses 66,67 . Moreover, injury-induced mediators, such as IL-1 and C5a may affect IFN-γ production and differentiation of Th1 cells. In general, the primary damage caused due to rejection may provoke a greater adaptive alloimmune response through speci c mediators, thereby inducing T cells to produce increased IFN-γ and OAT-induced vasculopathy-related proteins, which ultimately leads to vasculopathy. In this study, we observed that administering PAW could reduce the plasma IFN-γ concentration in OAT-ACI/NKyo rats to almost similar levels as that of control ACI/NKyo rats. Although HMGB1 expression did not decrease to the baseline level, it was signi cantly lower than the levels in ROW administered OAT-ACI/NKyo rats. We speculate that PAW can effectively modulate the adaptive alloimmune response after OAT by modifying the expression of IFN-γ. However, since tissue damage and immune responses after OAT are caused by multiple immune factors, administering PAW can only partially reduce HMGB1 production in OAT-ACI/NKyo rats. In the future, we consider using PAW in combination with existing clinical drugs to develop effective therapeutic strategies for OAT-induced vasculopathy.
In this study, although we only explored the effects of PAW on OAT-induced vasculopathy and obtained surprising results, we continue to study its detailed mechanism of action. Since drinking water is a natural activity that must be carried out in the life of human beings, if drinking PAW only as a treatment method, it will inevitably not be convinced by most people. Therefore, without deviating from the current focus on prevention and treatment of OAT-induced vasculopathy, we are initiating a study to elucidate the synergistic effects of PAW on anti-rejection and immunomodulatory agents, and analyze whether the use of PAW in combination with existing drugs can achieve better therapeutic effects than current treatment modalities. Overall, this study indicated that daily consumption of PAW might decrease the progress of vasculopathy in OAT-recipient ACI/NKyo rats by promoting the inhibition collagen accumulation, SMC and broblast in ltration, and Tc and Th cell responses in the vessel walls. Furthermore, the data showed reduced activation of T lymphocytes, B lymphocytes, plasma cells, and macrophages in the spleen of PAW administered OAT-recipient ACI/NKyo rats compared to ROW administered OAT-recipient ACI/NKyo rats. Finally, we observed higher mobilization and levels of circulating EPCs in PAW-administered OATrecipient ACI/NKyo rats compared to control group. Therefore, this study highlights the potential therapeutic roles of PAW in OAT-induced vasculopathy.

Animals
A statement con rming the study was carried out in compliance with the ARRIVE guidelines. All animals were treated according to the protocols approved by the Institutional Animal Care Committee of the National Defense Medical Center (certi cate Nos. IACUC-20-275). Experimental procedures and animal care conformed to the "Guide for the Care and Use of Laboratory Animals" published by the U.S. National Institutes of Health (NIH Publication No. 85 − 23, revised 1996).
Full Methods are available in the supplementary of the paper.