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.
Table 1
Comparison of Biochemical Parameters in Experimental ACI//NKyo Rats.
| Control ACI/NKyo (non-OAT) | | OAT + ROW (PVG/Seac to ACI/NKyo) | | OAT + PAW (PVG/Seac to ACI/NKyo) |
| baseline | 90 days | | baseline | 90 days | | baseline | 90 days |
Body weight (g) | 240.3 ± 18.6 | 350.1 ± 16.7 | 220.9 ± 12.8 | 332.5 ± 15.6 | 234.6 ± 10.2 | 365.7 ± 13.4 |
BUN (mg/dL) | 26.5 ± 5.6 | 30.0 ± 5.9 | 26.8 ± 9.0 | 29.5 ± 6.1 | 25.4 ± 7.1 | 29.8 ± 6.8 |
Creatinine (mg/dL) | 0.6 ± 0.1 | 0.7 ± 0.2 | 0.8 ± 0.5 | 0.8 ± 0.3 | 0.9 ± 0.3 | 0.9 ± 0.2 |
ALT (IU/L) | 19.9 ± 6.7 | 22.8 ± 9.1 | 21.3 ± 6.5 | 23.5 ± 9.1 | 25.4 ± 3.8 | 20.6 ± 1.9 |
AST (IU/L) | 30.5 ± 6.1 | 32.5 ± 8.1 | 30.5 ± 3.6 | 30.6 ± 8.1 | 29.8 ± 9.9 | 30.5 ± 9.1 |
OAT, orthotopic aortic transplantation; BW, body weight; BUN, blood urea nitrogen; ALT, alanine transaminase; AST, aspartate transaminase; Values are represented as mean ± SD. ap < 0.05 compared with baseline of the same group; bp < 0.05 compared with Control ACI/NKyo (non-OAT) group at the same time point; cp < 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. |
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 quantifications 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 calcified 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 significantly 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 calcified plaque formation.
Vasculopathy has been implicated in the development of collagen accumulation 16. Therefore, picro-sirius red staining was performed for visualizing collagen. As shown in Fig. 1C, normal thoracic aortas from PVG/Seac rats presented intact, thick collagen fibers (weak orange to red) and an equal distribution of fine collagen fibers (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 fine collagen fibers being piled up with a small amount of messy, thick collagen fibers. Moreover, the thick collagen fibers in the original vessel walls became inconsistent in thickness. However, in the OAT with PAW group, the disordered distribution and accumulation of fine and thick collagen fibers indeed improved significantly 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 fibroblasts 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. 2A).The OAT PVG/Seac thoracic aorta sections at day 90 showed marked accumulation of SMCs and fibroblasts in hyperplasia area on the luminal surface in ROW administered group compared to sections of the PAW administered group. CD4- and CD8-positive T cells are subtypes of lymphocytes. CD4-positive T cells are MHC class II-restricted and CD8-positive T cells are a critical subpopulation of regulatory T-lymphocytes involved in MHC class I-restricted interactions, and hence they are important mediators of adaptive immunity. Infiltrated CD11b positive macrophages also play critical roles in the process of chronic allograft vasculopathy. Therefore, immunohistochemical staining was performed on transplanted aorta sections to identify the immune reaction in the vessel using antibodies against CD4, CD8, and CD11b (Fig. 2B). The thoracic aorta sections from the OAT PVG/Seac ROW administered group at day 90 showed marked infiltration of CD4-, CD8-, and CD11b-positive cells. However, T lymphocytes and macrophages were accumulated less in the vessel wall in PAW administered group. These results indicated that relatively minimal adaptive immune reactions occurred in the vessel wall of the PAW administered group, resulting in reduced T lymphocyte and macrophage infiltration.
Administration of PAW results in lower plasma concentration of inflammatory factors and cytokines in OAT ACI/NKyo rats
As shown in Table 2, inflammation-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 significantly 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 inflammation and tissue damage 18 and HMGB1 is involved in the chronic rejection of cardiac allograft vasculopathy 19. As shown in Table 2, the CRP levels were increased after 90 days of OAT compared to the baseline in ROW (163.5 ± 22.5 mg/dL) or PAW (72.1 ± 5.4 mg/dL) plus OAT groups. Nevertheless, after OAT, PAW administered rats still had relatively low levels of CRP compared to the rats administered with ROW. Similar to CRP levels, the HMGB1 expression was lower in rats in the PAW group than that in ROW group. Additionally, the function of EPCs is related to the occurrence of OAT-induced vasculopathy 20. However, SDF-1α is involved in the homing and recruitment of EPCs, and TGF-β1 negatively regulates EPC function 21 following OAT. Regardless of whether the rats received ROW or PAW, OAT increased plasma SDF-1α and TGF-β1 levels. Especially, administration of PAW resulted in a significant SDF-1α increase (1211.4 ± 130.2 pg/mL) and TGF-β1 decrease (129.7 ± 54.1 ng/mL) at day 90 compared to that of the OAT plus ROW group (432.6 ± 100.4 pg/mL for SDF-1α and 378.1 ± 55.9 ng/mL for TGF-β1). IFN-γ mediates transplant vasculopathy through CD8+ or CD4+ T lymphocyte-associated injury of vascular endothelial cells. Furthermore, cytokines such as IL-2 cause a reversible insult to the endothelium around the time of transplantation 22. Administration of PAW may significantly decrease IFN-γ (3.5 ± 1.2 pg/mL) and IL-2 (352.4 ± 95.7 pg/mL) production in ACI/NKyo rats after OAT for 90 days compared to ROW administered group. Moreover IFN-γ expression almost reaches the basal level in PAW administered OAT ACI/NKyo rats. Based on these results, we predict that PAW may regulate alloimmunity and nonimmunity factors in an appropriate situation.
Table 2
Comparison of OAT-induced Vasculopathy-related Proteins in ACI//NKyo Rats.
| Control ACI/NKyo (non-OAT) | | OAT + ROW (PVG/Seac to ACI/NKyo) | | OAT + PAW (PVG/Seac to ACI/NKyo) |
| baseline | 90 days | baseline | 90 days | baseline | 90 days |
LDH (IU/L) | 900.5 ± 99.5 | 895.9 ± 125.6 | 854.6 ± 152.5 | 1888.78 ± 323.0ab | 802.3 ± 152.0 | 968.8 ± 116.4c |
CRP (mg/dL) | 40.2 ± 13.5 | 39.6 ± 9.1 | 39.4 ± 9.8 | 163.5 ± 22.5ab | 26.5 ± 8.1 | 72.1 ± 5.4abc |
HMGB1 (ng/mL) | 2.7 ± 1.4 | 2.8 ± 1.3 | 3.4 ± 1.5 | 98.7 ± 19.7ab | 2.5 ± 1.4 | 55.4 ± 13.1abc |
SDF-1α (pg/mL) | 200.3 ± 22.6 | 196.5 ± 52.1 | 220.1 ± 41.5 | 432.6 ± 100.4ab | 241.3 ± 54.1 | 1211.4 ± 130.2abc |
TGF-β1 (ng/mL) | 66.3 ± 21.2 | 78.9 ± 25.4 | 72.4 ± 19.7 | 378.1 ± 55.9ab | 72.1 ± 22.0 | 129.7 ± 54.1abc |
INF-γ (pg/mL) | 2.5 ± 0.8 | 3.1 ± 0.9 | 1.5 ± 0.7 | 18.7 ± 5.3ab | 1.9 ± 0.8 | 3.5 ± 1.2c |
IL-2 (pg/mL) | 45.7 ± 13.5 | 56.8 ± 18.9 | 53.7 ± 21.5 | 899.4 ± 102.4ab | 45.2 ± 21.0 | 352.4 ± 95.7abc |
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. ap < 0.05 compared with baseline of the same group; bp < 0.05 compared with control ACI/NKyo (non-OAT) group at the same time point; cp < 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 significantly 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 infiltrated 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 cell-mediated 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 and may induce endothelial-mesenchymal transition (endo-MT) to ameliorate OAT-induced vasculopathy. INF-γ expression plays an important role in the progression of OAT-induced vasculopathy 27. Therefore, following OAT surgery, the population of endogenous EPCs (defined as CD133+/CD34+/VEGF+ cells) and SMPCs (defined as CD133+/αSMA+/ CD34− cells) were quantified by flow cytometry, to compare the difference in levels of these cells between ROW and PAW administered ACI/NKyo rats. The results showed a significant 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 significantly 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 flow 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 fibronectin-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 efficient differentiation of MNCs. After 21 days of continuous culture, EPCs had cobblestone-like morphology similar to that of mature endothelial cells and were confluent 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.