The active form of vitamin D3, 1,25-dihydroxyvitamin D3 (1,25[OH]2D3), is a secosteroid hormone that regulates calcium and bone metabolism, controls cell proliferation and differentiation, and exerts immunoregulatory activities (1–3). The biologic effects of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR), a member of the superfamily of nuclear hormone receptors functioning as a ligand-activated transcription factor that binds to specific vitamin D-responsive elements in target genes and ultimately influences their rate of RNA polymerase II-mediated transcription (4).
Dendritic cells (DC) and T cells are key targets of VDR agonists. The immunomodulatory properties of VDR agonists enhance regulatory T cells, lead to selective inhibition of T helper (Th) type 1 cells and, under appropriate conditions, may favor a deviation to the Th2 pathway (5). These effects could be, in part, a consequence of direct T-cell targeting by VDR agonists, in particular by means of direct inhibition of Th1-type cytokines such as interferon (IFN)-γ and interleukin (IL)-2, but modulation of DC functions such as decrease of co-stimulatory molecules CD40, CD80, and CD86 expression; inhibition of IL-12; and enhancement of IL-10 production (6–8) certainly play an important role in induction of DC with tolerogenic properties able to shape the development of T-cell responses. The capacity of VDR agonists to target DC and T cells is mediated by VDR expression in both cell types and by the presence of common targets in their signal transduction pathways, such as the nuclear factor-κB that is decreased in DC (9) and in T cells (10).
These immunoregulatory activities of VDR agonists have been demonstrated in models of allograft rejection by oral administration directly to the recipient (11) or by adoptive transfer of in vitro-treated DC (12), and have been translated into effective immunologic intervention in a variety of graft rejection models, both acute and chronic (13, 14). VDR agonists can significantly prolong allograft survival in heart (15–17), kidney (18, 19), liver (20), pancreatic islets (11, 21–23), skin (24), and small bowel allografts (16). In most experimental models, the acute rejection has been further delayed by combining VDR agonists with a suboptimal dose of cyclosporine A (CsA) or other immunosuppressive agents (13). Interestingly, VDR agonists can inhibit, in association with low doses of CsA, not only acute but also chronic allograft rejection, as documented by inhibition of adventitial inflammation and intimal hyperplasia in rat aortic allografts (25).
VDR agonists have widespread clinical application, notably in the treatment of osteoporosis, secondary hyperparathyroidism, and psoriasis (26), but hypercalcemia is a dose-limiting effect that prevents sustained systemic administration. To overcome this limitation, a number of 1,25(OH)2D3 analogues, with a wider therapeutic window than 1,25(OH)2D3 itself, have been synthesized and shown to be effective in experimental models of allograft rejection (13). In the present study, we report the capacity of a nonhypercalcemic VDR agonist, BXL-628, to inhibit, as a monotherapy, acute and chronic allograft rejection, leading to markedly reduced intimal hyperplasia associated with significantly decreased expression of several muscle genes in the transplanted aorta.
MATERIALS AND METHODS
Mice and Treatments
C57BL/6 and BALB/c mice were purchased from Charles River Breeding Laboratories (Calco, Italy). Mice were kept under specific pathogen-free conditions. All animal studies were approved by the institutional review board. Calcitriol and the calcitriol analogue 1-α-fluoro-25-hydroxy-16,23E-diene-26,27-bishomo-20-epi-cholecalciferol (BXL-628) were provided by Dr. Milan Uskokovic (BioXell, Inc., Nutley, NJ). Dexamethasone was purchased from Sigma Chemical Co. (St. Louis, MO). BXL-628 was administered daily orally in a volume of 100 μL of vehicle (Miglyol 812) at a dose of 30 μg/kg per day starting the day before transplantation, and the treatment was continued until day 30 posttransplantation or until rejection. Calcitriol was administered 3 days per week orally at a dose of 5 μg/kg per day starting the day before transplantation, and the treatment was continued until day 30 posttransplantation or until rejection. Vehicle alone (Miglyol 812) was used as control. Dexamethasone was administered orally at 0.05 mg/kg or 0.25 mg/kg 5 days per week from day −1 to day 30. Treatment protocols are summarized in Table 1. Serum calcium levels were measured with a commercially available colorimetric assay (Sigma) according to the manufacturer's instructions.
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TABLE 1: Treatment protocols tested in acute and chronic allograft models
Vascularized Heart Transplantation
Heterotopic abdominal cardiac transplants in mice were performed between C57BL/6 donors and BALB/c recipients as described (27). Briefly, the aorta and vena cava of recipient mice were separated between the renal vessels and the bifurcation of the iliacs. The ascending aorta of the donor heart was connected by means of end-to-side anastomosis with the recipient aorta. A similar anastomosis was also created between the recipient vena cava and the superior vena cava of the donor heart. The total ischemic time averaged 15 min. Heterotopic cardiac survival was monitored by direct palpation of the heart beat through the abdominal wall.
Aortic Transplantation
Aortic transplantations were performed between C57BL/6 donors and BALB/c recipients, as described (28). A segment of donor dorsal aorta, approximately 7 mm long, was used to replace a segment of similar length in the abdominal aorta of the recipient. The implanted graft was examined to confirm median positioning and absence of kinking at the anastomotic sites. Graft patency was confirmed by the presence of pulsation in the graft and in the proximal and distal adjacent segments of native aorta. Using this protocol, the mice recovered from anesthesia a few minutes after administration of anesthetic was discontinued. No antibiotics were used. Motor and sensory functions of hind limbs were assessed immediately after recovery.
Determination of the Percentage of Luminal Reduction
Segments of aorta allografts were snap-frozen in Tissue Tek (Miles Laboratories, Elkhart, IN) and stored at −80°C. Luminal reduction was assessed 60 days posttransplantation in 5-μm-thick transverse cryostatic sections of the transplanted aorta stained with hematoxylin-eosin. To quantify the percentage of luminal reduction, histologic images were acquired with a Leica DC camera (Leica IM 1000 image manager) and evaluated using Leica Qwin software. The percentage of luminal reduction was defined according to the following formula: area bounded by the internal elastic lamina−area of the lumen×100÷the area bounded by the internal elastic lamina.
Immunohistochemistry
Segments of aorta allografts were snap-frozen in Tissue Tek (Miles Laboratories) and stored at −80°C. Frozen section (5-μm-thick) were air-dried and then fixed in acetone for 10 min. Endogenous peroxidase activity was blocked with 2% hydrogen peroxide and 0.1% sodium azide in cold Tris-buffered saline. Endogenous biotin was blocked by incubation with an avidin solution mixed in 1% bovine serum albumin in phosphate-buffered saline for 15 min and followed by a biotin solution mixed in 1% bovine serum albumin in phosphate-buffered saline for 15 min (Vector Laboratories, Ltd., Burlingame, CA). Sections were stained with biotinylated monoclonal antibodies against CD4, CD8, CD11b (all purchased from PharMingen, San Diego, CA), or CD11c (N418; American Type Culture Collection, Manassas, VA), followed by streptavidin-peroxidase conjugate. 3-Amino-9-ethylcarbazole (DAKO, Carpinteria, CA) was used as chromogen, and hematoxylin was used as a counterstain.
Microarray Analysis
The transplanted aortas were harvested, immediately placed into RNAlater reagent (Sigma), and stored at 4°C until processing. Tissue homogenization was performed by using a Wheaton-33 glass Tenbroeck type homogenizer in RNA isolation buffer and RNA was extracted using the Qiagen RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany). CodeLink Uniset Mouse 20k I BioArrays were purchased from Amersham Biosciences GE Healthcare (Piscataway, NJ). Total RNA (500 ng per sample) was copied into cDNA followed by in vitro biotin-labeled cRNA transcription according to the CodeLink Gene expression System protocol. The CodeLink BioArrays were hybridized as recommended, with a total of 10 μg of biotin-labeled cRNA, for 18 hr at 37°C in a shaking oven. All subsequent steps were performed as recommended by the manufacturer. Signal intensities were measured by an Axon 4000B scanner using Genepix 4.0 software (Axon Instruments, Inc., Union City, CA) according to the manufacturer's recommended settings (10-μm pixel size). The experimental design included replicate slides for every sample. The images were feature extracted using the CodeLink Analysis Software version 4.0. Feature-extracted data were imported into the GeneSpring Software package version 7.0 (Silicon Genetics, Redwood City, CA). Differentially expressed genes between BXL-628 or Miglyol-treated groups were compared by fold increase or decrease, with Gene Ontology-defined terms (www.geneontology.org), to analyze gene products in terms of their associated biologic processes, cellular components, and molecular functions.
Statistical Analysis
Numerical data are expressed as mean±SEM. Statistical analysis was performed using a nonparametric Student's t test.
RESULTS
Inhibition of Vascularized Heart Allograft Rejection by Treatment with BXL-628
To determine the capacity to inhibit acute allograft rejection in the vascularized heart transplantation model, BXL-628 or calcitriol was administered to graft recipients according to the protocol summarized in Table 1. Vehicle alone (Miglyol 812) was used as control. Results in Figure 1 show that administration of BXL-628 as a monotherapy to mice receiving fully mismatched vascularized cardiac allografts resulted in a statistically significant prolongation of graft survival compared with administration of vehicle alone (mean graft survival time, 21.8±2.1 vs. 9±0.9 days; P=0.002). Prolongation of graft survival was more sustained after treatment with BXL-628 compared with calcitriol (Fig. 1). Dexamethasone treatment was not tested in this model. These results show the in vivo immunosuppressive effect of BXL-628 in the context of an acute model of allotransplantation and demonstrate that its efficacy is superior to calcitriol.
FIGURE 1.:
Inhibition of vascularized heart allograft rejection by treatment with BXL-628. Graft survival time (GST) of vascularized C57BL/6 hearts transplanted into BALB/c recipients treated orally from days −1 to 30 or until rejection with vehicle alone (Miglyol 812) daily 5 days per week, BXL-628 daily 5 days per week, or calcitriol 3 days per week.
Inhibition of Intimal Cell Proliferation in Aortic Allografts by BXL-628 Treatment
The mouse aortic allograft model provides an animal model of immune-mediated vascular intimal thickening, which is similar to the vascular lesions of human chronic allograft rejection. Preliminary experiments have demonstrated VDR expression in aortic cells by real-time reverse-transcriptase polymerase chain reaction (data not shown), consistent with the VDR expression found in vascular smooth muscle cells (29). BXL-628, calcitriol, or dexamethasone was administered as monotherapy to mice receiving fully mismatched aortic allografts according to the protocol described in Table 1. At the end of the 30-day treatment, serum calcium levels were within the normal range after administration of BXL-628 or calcitriol (Table 2).
TABLE 2: BXL-628 is superior to calcitriol and dexamethasone in blocking neointima formation in aorta allografts
Mice receiving BXL-628, calcitriol, dexamethasone, or vehicle alone were sacrificed at day 60 posttransplant and graft luminal narrowing was determined on histologic sections, after hematoxylin-eosin staining, using Leica imaging software. At this time point, 30 days after treatment withdrawal, vehicle-treated mice had progressed to a marked proliferation of intimal cells with a consequent reduction of the aortic lumen. This was significantly reduced by dexamethasone treatment, but to a higher extent by calcitriol and even more by BXL-628, which led to an approximately 80% reduction of intimal hyperplasia compared with vehicle controls, even after 30 days of treatment withdrawal (Table 2). The effect of BXL-628 was significantly superior to dexamethasone, administered at 0.05 mg/kg (P=0.0079) or 0.25 mg/kg (P=0.0043). No difference could be seen between the two doses of dexamethasone administered, possibly suggesting that the lower dose tested had already induced a maximal effect. Representative histologic findings are shown in Figure 2. Thus, BXL-628 monotherapy significantly inhibits vascular intimal thickening in the aortic allograft model without increasing serum calcium levels.
FIGURE 2.:
Inhibition of intimal cell proliferation in aortic allografts by BXL-628 treatment. Histopathologic evaluation, 60 days after transplantation of C57BL/6 into BALB/c aortic grafts, of allografts from recipient mice treated with vehicle, calcitriol, BXL-628, or dexamethasone from day −1 to day 30, according to the treatment protocols outlined in
Table 1. Snap-frozen sections were stained with hematoxylin-eosin. Aortic allograft from vehicle-treated mice show a well-established intimal thickening (approximately 70% luminal reduction) that is markedly decreased in BXL-628-treated mice (magnification ×100).
Inhibition of Leukocyte Recruitment in Aortic Allografts by BXL-628 Treatment
In the same aortic allografts, immunohistologic analysis was performed to evaluate the degree of leukocyte infiltration into the graft. Results in Figure 3 demonstrate a considerable infiltration of CD11b+ macrophages and CD11c+ dendritic cells, in particular, in the adventitia of the transplanted aorta in vehicle-treated mice. T-cell infiltration (CD4+ or CD8+) was much less apparent. The extent of infiltration was quantitated by cell counting with Leica imaging software, and the data demonstrate that BXL-628 is more active than calcitriol and the high dose of dexamethasone (0.25 mg/kg 5 days per week) in inhibiting leukocyte infiltration, leading to a significant reduction into the grafted aorta of both macrophages and dendritic cells.
FIGURE 3.:
Quantitation of leukocyte recruitment inhibition into aortic allografts. Immunohistologic analysis with antibodies to the indicated cell markers was performed to evaluate the degree of leukocyte infiltration into the graft. The extent of cell infiltration was quantitated by cell counting with Leica imaging software (see
Materials and Methods) 60 days after transplantation of aortic grafts in recipient BALB/c mice treated with vehicle, calcitriol, BXL-628, or dexamethasone from day −1 to day 30, according to the treatment protocols outlined in
Table 1.
Effect of BXL-628 Treatment on Muscle-Related Gene Expression in Aortic Allografts
A series of microarray experiments were conducted to investigate possible mechanisms of action of BXL-628, and a full report will be published separately. In one set of experiments, aortic allografts were harvested after a 30-day treatment with Miglyol alone or containing 30 μg/kg BXL-628, and RNA was isolated. Each sample was hybridized in duplicate on microarrays containing probes for approximately 20,000 well-characterized mouse genes. The data were normalized and filtered for quality flags. The gene expression patterns of aortic allografts from vehicle- and BXL-628–treated mice were clearly distinguishable, with over 1,000 genes showing more than a 1.5-fold change. Comparison of the differentially expressed genes with established groups of genes as described by the Gene Ontology project shows that the gene cluster most differentially expressed (P=2.68×10−8) has been annotated as Gene Ontology biological process: Muscle development.
Within this group of 43 genes annotated to be involved in muscle development, 21 are not expressed (data not shown), 8 are not significantly changed in expression, and 14 are significantly decreased (Table 3). Among the genes significantly decreased, several have been implicated in stretch-induced smooth muscle development, such as desmin, α-actin, transgelin, and tropomyosin (30). In addition, several myosin genes are significantly decreased. These results are consistent with the marked inhibition of intimal hyperplasia observed by histologic analysis and demonstrate a pronounced effect of BXL-628 treatment on smooth muscle cells.
TABLE 3: Muscle development genes in aortic allografts from vehicle and BXL-628-treated recipient mice
DISCUSSION
Chronic rejection is not prevented by current immunosuppressive protocols (31). Agents able to inhibit chronic rejection, and potentially able to promote transplantation tolerance, would thus fill an important unmet medical need. Results in this study suggest the clinical applicability of VDR agonists, such as BXL-628, in the long-term management of allograft rejection, with the aim of preventing chronic graft rejection without inducing major adverse effects.
The induction of tolerogenic DC by VDR agonists, which leads to an enhanced number of CD4+CD25+ regulatory T cells in vivo (11, 32), is likely to play an important role in controlling graft rejection, both acute and chronic, and in favoring the establishment of transplantation tolerance (3, 33, 34). The induction of tolerogenic DC could indeed represent a therapeutic strategy promoting tolerance to allografts (34), and this mechanism may be shared by several immunosuppressive and anti-inflammatory agents used to control allograft rejection (35).
In addition, the direct effects of VDR agonists on T cells, in particular, the inhibition of IL-2 and IFN-γ production, could play a role in inhibiting graft rejection. 1,25(OH)2D3 inhibits IL-2 secretion by impairing the formation of the transcription factor complex NF-AT (36) and IFN-γ through interaction of the ligand-bound VDR complex with a vitamin D response element in the promoter region of the cytokine (37). A combination of 1,25(OH)2D3 and low-dose CsA inhibited the expression of IL-2 and IL-12, and increased significantly IL-10 expression levels in kidney allografts (19). Additional mechanisms could rely on the capacity of 1,25(OH)2D3 to significantly reduce bioactive renal transforming growth factor (TGF)-β1 by interacting with Smad proteins, important regulators of TGF-β signal transduction (38). Because TGF-β has a pronounced profibrotic activity, its decrease in the kidney tissue may inhibit the evolution of chronic rejection in kidney transplants.
Although the prevention of leukocyte infiltration into the adventitia is probably attributable to the immunomodulatory properties of BXL-628, shared by 1,25(OH)2D3 and other VDR agonists, the inhibition of intimal cell proliferation, both endothelial and smooth muscle cells, is likely induced by their capacity to regulate cell growth. Inhibition of intimal thickening in rat aortic allografts was also induced by the VDR agonist MC1288 combined with CsA, but the doses used caused hypercalcemia and weight loss (25). MC1288 also reduced clinical and histologic signs of chronic graft rejection in rat kidney allografts, but the chronic allograft damage index was significantly reduced only in recipients treated with both MC1288 and CsA (18). Conversely, in our experiments, treatment with BXL-628, as a monotherapy, was able to reduce intimal hyperplasia by over 80%, compared with vehicle-treated controls, without inducing hypercalcemia, the major side effect of VDR agonists.
The muscle is a nontraditional target of VDR agonists (39), as shown by the aberrantly elevated expression of myogenic differentiation markers and myoregulatory transcription factors in VDR-deficient mice (40). The inhibition by BXL-628 treatment of most expressed genes related to muscle development in aortic allografts shows that smooth muscle cells are important targets of this VDR agonist, consistent with the in vitro inhibition of desmin induced by BXL-628 in stressed smooth muscle bladder cells (41). In addition to desmin, the present results show that several other smooth muscle-related genes, such as transgelin, tropomyosin, α-actin, and myosin heavy chain 11, are significantly decreased in aortic allografts after BXL-628 treatment. Intriguingly, the gene encoding the myocyte enhancer factor 2C, which was found significantly inhibited in aortic allografts by BXL-628 treatment, has been shown to interact with steroid receptor coactivator family members (42), which are required for calcitriol signaling to the nucleus in muscle cells (43).
On the basis of the available preclinical evidence of a protolerogenic effect and a reduced incidence of chronic rejection, VDR agonists could be added to standard immunosuppressive regimens in the treatment of allograft rejection. Additive and even synergistic effects have been observed between 1,25(OH)2D3 or its analogues and immunosuppressive agents, in particular, CsA, tacrolimus, and sirolimus (44). These effects have been confirmed in models of graft rejection (13), making VDR agonists potentially interesting as dose-reducing agents for conventional immunosuppressive drugs in clinical transplantation. In this respect, BXL-628 could represent a suitable candidate, given its capacity to markedly inhibit neointimal proliferation and leukocyte infiltration at nonhypercalcemic doses.
In a retrospective study, patients receiving 1,25(OH)2D3, in addition to standard immunosuppressive treatment, showed decelerated renal graft loss (45), providing further evidence for the clinical applicability of VDR agonists to inhibit chronic graft rejection. Another positive feature of adding VDR agonists to standard immunosuppressive regimens is their protective effect on bone loss (46). Importantly, VDR agonists do not appear to increase opportunistic infections (47), a clinically relevant side effect induced by antirejection drugs such as calcineurin inhibitors and glucocorticoids, and actually can directly induce antimicrobial peptide gene expression (48).
CONCLUSION
BXL-628 inhibits, as a monotherapy in mouse models, both acute and chronic graft rejection, suggesting that its addition to clinical immunosuppressive regimens may have a positive impact on long-term graft function.
REFERENCES
1.Deluca HF, Cantorna MT. Vitamin D: Its role and uses in immunology.
FASEB J 2001; 15(14): 2579.
2.Mathieu C, Adorini L. The coming of age of 1,25-dihydroxyvitamin D(3) analogs as immunomodulatory agents.
Trends Mol Med 2002; 8(4): 174.
3.Griffin MD, Xing N, Kumar R. Vitamin D and its analogs as regulators of immune activation and antigen presentation.
Annu Rev Nutr 2003; 23: 117.
4.Carlberg C, Polly P. Gene regulation by vitamin D3.
Crit Rev Eukaryot Gene Expr 1998; 8(1): 19.
5.Adorini L, Penna G, Giarratana N, et al. Tolerogenic dendritic cells induced by vitamin D receptor ligands enhance regulatory T cells inhibiting allograft rejection and autoimmune diseases.
J Cell Biochem 2003; 88(2): 227.
6.Penna G, Adorini L. 1,25-dihydroxyvitamin D3 inhibits differentiation, maturation, activation and survival of dendritic cells leading to impaired alloreactive T cell activation.
J Immunol 2000; 164: 2405.
7.Piemonti L, Monti P, Sironi M, et al. Vitamin D3 affects differentiation, maturation, and function of human monocyte-derived dendritic cells.
J Immunol 2000; 164(9): 4443.
8.Griffin MD, Lutz WH, Phan VA, et al. Potent inhibition of dendritic cell differentiation and maturation by
vitamin D analogs.
Biochem Biophys Res Commun 2000; 270(3): 701.
9.Dong X, Craig TA, Xing N, et al. Direct transcriptional regulation of RelB by 1alpha,25-dihydroxyvitamin D3 and its analogs: Physiologic and therapeutic implications for dendritic cell function.
J Biol Chem 2003; 278: 49378.
10.Barrat FJ, Cua DJ, Boonstra A, et al. In vitro generation of interleukin 10-producing regulatory CD4(+) T cells is induced by immunosuppressive drugs and inhibited by T helper type 1 (Th1)- and Th2-inducing cytokines.
J Exp Med 2002; 195(5): 603.
11.Gregori S, Casorati M, Amuchastegui S, et al. Regulatory T cells induced by 1alpha,25-dihydroxyvitamin D3 and mycophenolate mofetil treatment mediate transplantation tolerance.
J Immunol 2001; 167: 1945.
12.Griffin MD, Lutz W, Phan VA, et al. Dendritic cell modulation by 1alpha,25 dihydroxyvitamin D3 and its analogs: A vitamin D receptor-dependent pathway that promotes a persistent state of immaturity in vitro and in vivo.
Proc Natl Acad Sci USA 2001; 22: 22.
13.Adorini L. 1,25-Dihydroxyvitamin D3 analogs as potential therapies in transplantation.
Curr Opin Investig Drugs 2002; 3(10): 1458.
14.Becker BN, Hullett DA, O'Herrin JK, et al. Vitamin D as immunomodulatory therapy for kidney transplantation.
Transplantation 2002; 74(8): 1204.
15.Lemire JM, Archer DC, Khulkarni A, et al. Prolongation of the survival of murine cardiac allografts by the vitamin D3 analogue 1,25-dihydroxy-delta 16-cholecalciferol.
Transplantation 1992; 54(4): 762.
16.Johnsson C, Tufveson G. MC 1288: A vitamin D analogue with immunosuppressive effects on heart and small bowel grafts.
Transpl Int 1994; 7: 392.
17.Hullett DA, Cantorna MT, Redaelli C, et al. Prolongation of allograft survival by 1,25-dihydroxyvitamin D3.
Transplantation 1998; 66(7): 824.
18.Kallio E, Hayry P, Pakkala S. MC1288, a vitamin D analogue, reduces short- and long-term renal allograft rejection in the rat.
Transplant Proc 1996; 28(6): 3113.
19.Redaelli CA, Wagner M, Gunter-Duwe D, et al. 1Alpha,25-dihydroxyvitamin D3 shows strong and additive immunomodulatory effects with cyclosporine A in rat renal allotransplants.
Kidney Int 2002; 61(1): 288.
20.Redaelli CA, Wagner M, Tien YH, et al. 1Alpha,25-dihydroxycholecalciferol reduces rejection and improves survival in rat liver allografts.
Hepatology 2001; 34(5): 926.
21.Casteels K, Waer M, Laureys J, et al. Prevention of autoimmune destruction of syngeneic islet grafts in spontaneously diabetic nonobese diabetic mice by a combination of a vitamin D3 analog and cyclosporine.
Transplantation 1998; 65(9): 1225.
22.van Etten E, Gysemans C, Verstuyf A, et al. Immunomodulatory properties of a 1,25(OH)(2) vitamin D(3) analog combined with IFNbeta in an animal model of syngeneic islet transplantation.
Transplant Proc 2001; 33(3): 2319.
23.Gysemans C, Waer M, Laureys J, et al. A combination of KH1060, a vitamin D(3) analogue, and cyclosporin prevents early graft failure and prolongs graft survival of xenogeneic islets in nonobese diabetic mice.
Transplant Proc 2001; 33(3): 2365.
24.Veyron P, Pamphile R, Binderup L, et al. Two novel vitamin D analogues, KH 1060 and CB 966, prolong skin allograft survival in mice.
Transpl Immunol 1993; 1(1): 72.
25.Raisanen-Sokolowski AK, Pakkala IS, Samila SP, et al. A vitamin D analog, MC1288, inhibits adventitial inflammation and suppresses intimal lesions in rat aortic allografts.
Transplantation 1997; 63(7): 936.
26.Pinette KV, Yee YK, Amegadzie BY, et al. Vitamin D receptor as a drug discovery target.
Mini Rev Med Chem 2003; 3(3): 193.
27.Corry RJ, Winn HJ, Russell PS. Primarily vascularized allografts of hearts in mice: The role of H-2D, H-2K, and non-H-2 antigens in rejection.
Transplantation 1973; 16(4): 343.
28.Sun H, Valdivia LA, Subbotin V, et al. Improved surgical technique for the establishment of a murine model of aortic transplantation.
Microsurgery 1998; 18(6): 368.
29.Merke J, Hofmann W, Goldschmidt D, et al. Demonstration of 1,25(OH)2 vitamin D3 receptors and actions in vascular smooth muscle cells in vitro.
Calcif Tissue Int 1987; 41(2): 112.
30.Albinsson S, Nordstrom I, Hellstrand P. Stretch of the vascular wall induces smooth muscle differentiation by promoting actin polymerization.
J Biol Chem 2004; 279(33): 34849.
31.Libby P, Pober JS. Chronic rejection.
Immunity 2001; 14(4): 387.
32.Gregori G, Giarratana N, Smiroldo S, et al. A 1alpha,25-dihydroxyvitamin D
3 analog enhances regulatory T cells and arrests autoimmune diabetes in NOD mice.
Diabetes 2002; 51: 1367.
33.Wood KJ, Sakaguchi S. Regulatory T cells in transplantation tolerance.
Nat Rev Immunol 2003; 3(3): 199.
34.Hackstein H, Thomson AW. Dendritic cells: Emerging pharmacological targets of immunosuppressive drugs.
Nat Rev Immunol 2004; 4(1): 24.
35.Adorini L, Giarratana N, Penna G. Pharmacological induction of tolerogenic dendritic cells and regulatory T cells.
Semin Immunol 2004; 16(2): 127.
36.Alroy I, Towers T, Freedman L. Transcriptional repression of the interleukin-2 gene by vitamin D3: Direct inhibition NFATp/AP-1 complex formation by a nuclear hormone receptor.
Mol Cell Biol 1995; 15: 5789.
37.Cippitelli M, Santoni A. Vitamin D3: A transcriptional modulator of the IFN-gamma gene.
Eur J Immunol 1998; 28: 3017.
38.Aschenbrenner JK, Sollinger HW, Becker BN, et al. 1,25-OH(2)D(3) alters the transforming growth factor beta signaling pathway in renal tissue.
J Surg Res 2001; 100(2): 171.
39.Demay M. Muscle: A nontraditional 1,25-dihydroxyvitamin D target tissue exhibiting classic hormone-dependent vitamin D receptor actions.
Endocrinology 2003; 144(12): 5135.
40.Endo I, Inoue D, Mitsui T, et al. Deletion of vitamin D receptor gene in mice results in abnormal skeletal muscle development with deregulated expression of myoregulatory transcription factors.
Endocrinology 2003; 144(12): 5138.
41.Crescioli C, Morelli A, Adorini L, et al. Human bladder as a novel target for vitamin D receptor ligands.
J Clin Endocrinol Metab 2005; 90(2): 962.
42.Lazaro JB, Bailey PJ, Lassar AB. Cyclin D-cdk4 activity modulates the subnuclear localization and interaction of MEF2 with SRC-family coactivators during skeletal muscle differentiation.
Genes Dev 2002; 16(14): 1792.
43.Buitrago C, Boland R, de Boland AR. The tyrosine kinase c-Src is required for 1,25(OH)2-vitamin D3 signalling to the nucleus in muscle cells.
Biochim Biophys Acta 2001; 1541(3): 179.
44.van Etten E, Branisteanu DD, Verstuyf A, et al. Analogs of 1,25-dihydroxyvitamin D3 as dose-reducing agents for classical immunosuppressants.
Transplantation 2000; 69(9): 1932.
45.O'Herrin JK, Hullett DA, Heisey DM, et al. A retrospective evaluation of 1,25-dihydroxyvitamin D(3) and its potential effects on renal allograft function.
Am J Nephrol 2002; 22(5–6): 515.
46.Stempfle HU, Werner C, Siebert U, et al. The role of tacrolimus (FK506)-based immunosuppression on bone mineral density and bone turnover after cardiac transplantation: A prospective, longitudinal, randomized, double-blind trial with calcitriol.
Transplantation 2002; 73(4): 547.
47.Cantorna MT, Hullett DA, Redaelli C, et al. 1,25-Dihydroxyvitamin D3 prolongs graft survival without compromising host resistance to infection or bone mineral density.
Transplantation 1998; 66(7): 828.
48.Wang TT, Nestel FP, Bourdeau V, et al. Cutting edge: 1,25-Dihydroxyvitamin D3 is a direct inducer of antimicrobial peptide gene expression.
J Immunol 2004; 173(5): 2909.
