Administration Of FK506 Research Articles

Dipeptidyl Peptidase 4 Inhibition Ameliorates Hypertensive Heart Failure via Suppression of Angiotensin-II-dependent Natrium Loading and Modulating NHE Expression

are widely used for drug delivery to target specific organs with enhanced vascular permeability. Since enhanced vascular permeability would be observed in myocarditis hearts due to severe inflammation, we hypothesized that immunosuppressive agent encapsulated with liposomes would enhance its therapeutic effects. In this study, we have prepared polyethylene glycol-modified liposomes encapsulating FK506 (liposomal FK506) (4109 nm) and examined its effects in rat model of experimental autoimmune myocarditis (EAM) in which Lewis rats were sensitized with porcine cardiac myosin. We could observe giant cells, which were quite similar to those in patients with GCM, in EAM hearts. Ex vivo fluorescent imaging revealed that intravenously administered nano-size particles accumulated in the myocarditis hearts. So we examined the tissue distribution of liposomal and free 3H-labeled FK506. We also examined the expression of cytokines in EAM hearts and found that some cytokines decreased more in liposomal FK506 administration. And liposomal FK506 significantly improved cardiac function at 21 days after sensitization. The immunosuppressive agent encapsulated with liposomes could be a novel therapy for GCM.

Periostin Cre-mediated Hypoxia-inducible Transfroming Growth Factor-β Receptor Deletion in Pulmonary Artery Prevents Pulmonary Arterial Hypertension in Mice

are widely used for drug delivery to target specific organs with enhanced vascular permeability. Since enhanced vascular permeability would be observed in myocarditis hearts due to severe inflammation, we hypothesized that immunosuppressive agent encapsulated with liposomes would enhance its therapeutic effects. In this study, we have prepared polyethylene glycol-modified liposomes encapsulating FK506 (liposomal FK506) (4109 nm) and examined its effects in rat model of experimental autoimmune myocarditis (EAM) in which Lewis rats were sensitized with porcine cardiac myosin. We could observe giant cells, which were quite similar to those in patients with GCM, in EAM hearts. Ex vivo fluorescent imaging revealed that intravenously administered nano-size particles accumulated in the myocarditis hearts. So we examined the tissue distribution of liposomal and free 3H-labeled FK506. We also examined the expression of cytokines in EAM hearts and found that some cytokines decreased more in liposomal FK506 administration. And liposomal FK506 significantly improved cardiac function at 21 days after sensitization. The immunosuppressive agent encapsulated with liposomes could be a novel therapy for GCM.

Calcineurin phosphatase as a negative regulator of fear memory in hippocampus: control on nuclear factor-κB signaling in consolidation and reconsolidation.

Protein phosphatases are important regulators of neural plasticity and memory. Some studies support that the Ca(2+) /calmodulin-dependent phosphatase calcineurin (CaN) is, on the one hand, a negative regulator of memory formation and, on the other hand, a positive regulator of memory extinction and reversal learning. However, the signaling mechanisms by which CaN exerts its action in such processes are not well understood. Previous findings support that CaN negatively regulate the nuclear factor kappaB (NF-κB) signaling pathway during extinction. Here, we have studied the role of CaN in contextual fear memory consolidation and reconsolidation in the hippocampus. We investigated the CaN control on the NF-κB signaling pathway, a key mechanism that regulates gene expression in memory processes. We found that post-training intrahippocampal administration of the CaN inhibitor FK506 enhanced memory retention one day but not two weeks after training. Accordingly, the inhibition of CaN by FK506 increased NF-κB activity in dorsal hippocampus. The administration of the NF-κB signaling pathway inhibitor sulfasalazine (SSZ) impeded the enhancing effect of FK506. In line with our findings in consolidation, FK506 administration before memory reactivation enhanced memory reconsolidation when tested one day after re-exposure to the training context. Strikingly, memory was also enhanced two weeks after training, suggesting that reinforcement during reconsolidation is more persistent than during consolidation. The coadministration of SSZ and FK506 blocked the enhancement effect in reconsolidation, suggesting that this facilitation is also dependent on the NF-κB signaling pathway. In summary, our results support a novel mechanism by which memory formation and reprocessing can be controlled by CaN regulation on NF-κB activity.

Initial Evidence for Functional Immune Modulation in Primate Recipients of Porcine Skin Grafts Following Conditioning With Human CD47 Transgenic Pig Hematopoietic Stem Cells.

Background: To inhibit phagocytosis of porcine cells by primate macrophages in tolerization protocols including mixed chimerism, we generated human CD47 transgenic swine on an α-1,3-galactosyltransferase null background (hCD47/GalT-KO). We report the initial results of hematopoietic stem (HSC) transplantation from this swine into baboons with respect to cell survival and immune response. Methods: Two baboons underwent a conditioning regimen consisting of non-myeloablative total body and thymic irradiation, T and B cell depletion, and infusion of HSC obtained by cytokine mobilization and leukapheresis of a CD47 transgenic GalT-KO swine. Immunosuppression consisted of clinical level administration of FK506 for the first 30 days and anti-CD154 antibody for the first 7 weeks following cell transplant. Recipients received challenge infusions of HSPC from the same donor swine 7 and 10 weeks following the initial infusion. Both baboons received split thickness skin grafts from the porcine donor on full thickness wound beds at 14 weeks without any additional immunosuppression. Results: Peripheral macrochimerism was detected following three cell transplants, with >10% levels observed at 24 hours declining over the next 3-7 days. Despite detectable macrochimerism no increase in anti-pig antibody levels or T-cell sensitization was observed prior to skin grafting. Both xenogeneic skin grafts developed extensive areas of bruising during the first week, while maintaining islands of apparently healthy tissue. These islands underwent expansion, reaching confluence over the entire wound bed in one recipient 6 weeks after graft placement and undergoing slow rejection over the following 5 weeks. In vitro measures of T cell responsiveness showed no evidence of sensitization to the xenografts prior to rejection. Conclusions: Assessment of the definitive role of hCD47 transgenic HSC transplantation in these results must await further ongoing studies of non-transgenic cell transplantation in similarly conditioned recipients. Nevertheless, the level and duration of chimerism using hCD47/GalT-KO mobilized HSC is very encouraging and may have been responsible for the greatly attenuated response against nonvascular components of xenogeneic skin grafts that we have observed in the absence of additional immunosuppression.

Casein Kinase II Inhibition Reverses Pain Hypersensitivity and Potentiated Spinal N-Methyl-d-aspartate Receptor Activity Caused by Calcineurin Inhibitor

Clinically used calcineurin inhibitors, including tacrolimus (FK506) and cyclosporine A, can induce calcineurin inhibitor-induced pain syndrome (CIPS), which is characterized as severe pain and pain hypersensitivity. Increased synaptic N-methyl-D-aspartate receptor (NMDAR) activity in the spinal dorsal horn plays a critical role in the development of CIPS. Casein kinase II (CK2), a serine/threonine protein kinase, can regulate synaptic NMDAR activity in the brain. In this study, we determined whether spinal CK2 is involved in increased NMDAR activity and pain hypersensitivity caused by systemic administration of FK506 in rats. FK506 treatment caused a large increase in the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) evoked by primary afferent stimulation and in the frequency of miniature EPSCs of spinal dorsal horn neurons. CK2 inhibition with either 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole (DRB) or 4,5,6,7-tetrabromobenzotriazole (TBB) completely normalized the amplitude of evoked NMDAR-EPSCs of dorsal horn neurons in FK506-treated rats. In addition, DRB or TBB significantly attenuated the amplitude of NMDAR currents elicited by puff application of N-methyl-D-aspartate to dorsal horn neurons in FK506-treated rats. Furthermore, treatment with DRB or TBB significantly reduced the frequency of miniature EPSCs of spinal dorsal horn neurons increased by FK506 treatment. In addition, intrathecal injection of DRB or TBB dose-dependently reversed tactile allodynia and mechanical hyperalgesia in FK506-treated rats. Collectively, our findings indicate that CK2 inhibition abrogates pain hypersensitivity and increased pre- and postsynaptic NMDAR activity in the spinal cord caused by calcineurin inhibitors. CK2 inhibitors may represent a new therapeutic option for the treatment of CIPS.

Calcineurin inhibitor induces pain hypersensitivity by potentiating pre‐ and postsynaptic NMDA receptor activity in spinal cords

Calcineurin inhibitors, such as cyclosporin A and tacrolimus (FK506), have played a pivotal role in the preservation of allograft function. However, these drugs can cause unexplained severe pain in patients, often referred to as calcineurin inhibitor-induced pain syndrome (CIPS). Although calcineurin can regulate NMDA receptor (NMDAR) activity, the causal relationship between spinal synaptic plasticity and CIPS remains unknown. In this study, we showed that systemic administration of FK506 (1.5 mg kg(-1) day(-1)) for 7 days in rats led to long-lasting nociceptive and mechanical hypersensitivity. Whole-cell patch-clamp recordings in spinal cord slices revealed that FK506 treatment caused a large increase in the amplitude of NMDAR-mediated excitatory postsynaptic currents (EPSCs) of dorsal horn neurons evoked by dorsal root stimulation. The amplitude of NMDAR currents elicited by puff NMDA application to dorsal horn neurons was also significantly greater in FK506-treated than in vehicle-treated rats. The frequency of spontaneous and miniature EPSCs in most dorsal horn neurons was profoundly increased in FK506-treated rats and was reduced by blocking NMDARs. Furthermore, blocking GluN2A or GluN2B subunits similarly reduced the amplitude of evoked EPSCs and the frequency of miniature EPSCs in dorsal horn neurons of FK506-treated rats. In addition, intrathecal injection of an NMDAR antagonist or systemic administration of memantine effectively reversed nociceptive and mechanical hypersensitivity in FK506-treated rats. Our findings indicate that calcineurin inhibition increases glutamate-mediated nociceptive input by potentiating presynaptic and postsynaptic NMDAR activity in spinal cords. NMDAR antagonists may represent a new therapeutic option for the treatment of CIPS.

Effects of FK-506 and CTLA4–Ig on nerve allografts in mice

The purpose of this study is to evaluate the effects of FK-506 and cytotoxic T lymphocyte-associated antigen-4 immunoglobulin fusion protein (CTLA4--Ig) on nerve allografts. Adult male inbred C3H mice (mean weight, 25 g) surgically received 8 mm posterior tibial nerve defects, and donor nerve allografts from donor male C57BL mice were implanted. The experimental animals were divided into five groups of 12 animals each that were distinguished from each other by administration of FK-506 and CTLA4--Ig, that is, isografts interposed in the gap between contralateral posterior tibial nerves (group A, positive control); allografts from C57BL mice implanted without administering any treatment (group B, negative control); allografts from C57BL mice implanted and FK-506 injections administered (group C); allografts from C57BL mice implanted and CTLA4--Ig injections administered (group D); and allografts from C57BL mice implanted and FK-506 and CTLA4--Ig injections administered (group E). Postoperative walking-track functional analysis and histomorphometric studies were then conducted. Data were statistically analysed using the Kruskal-Wallis test. Compared with the negative control (group B), mice treated with both FK-506 and CTLA4--Ig (group E) demonstrated better results at 3 weeks post operation. Similar values were observed in all groups, and there were no statistical differences at 6 weeks post operation. Our results demonstrate that a co-administration of FK-506 and CTLA4--Ig results in functional and histomorphometric recovery that is superior to that seen in the absence of these medications.

Tacrolimus (FK506) prevents early stages of ethanol induced hepatic fibrosis by targeting LARP6 dependent mechanism of collagen synthesis.

Tacrolimus (FK506) is a widely used immunosuppressive drug. Its effects on hepatic fibrosis have been controversial and attributed to immunosuppression. We show that in vitro FK506, inhibited synthesis of type I collagen polypeptides, without affecting expression of collagen mRNAs. In vivo, administration of FK506 at a dose of 4 mg/kg completely prevented development of alcohol/carbon tetrachloride induced liver fibrosis in rats. Activation of hepatic stellate cells (HSCs) was absent in the FK506 treated livers and expression of collagen α2(I) mRNA was at normal levels. Collagen α1(I) mRNA was increased in the FK506 treated livers, but this mRNA was not translated into α1(I) polypeptide. No significant inflammation was associated with the fibrosis model used. FK506 binding protein 3 (FKBP3) is one of cellular proteins which binds FK506 with high affinity. We discovered that FKBP3 interacts with LARP6 and LARP6 is the major regulator of translation and stability of collagen mRNAs. In the presence of FK506 the interaction between FKBP3 and LARP6 is weakened and so is the pull down of collagen mRNAs with FKBP3. We postulate that FK506 inactivates FKBP3 and that lack of interaction of LARP6 and FKBP3 results in aberrant translation of collagen mRNAs and prevention of fibrosis. This is the first report of such activity of FK506 and may renew the interest in using this drug to alleviate hepatic fibrosis.

Nerve Regeneration in Rat Limb Allografts

Successful nerve regeneration is critical to the functional success of composite tissue allografts. The present study was designed to characterize the effect of acute rejection on nerve regeneration and functional recovery in the setting of orthotopic limb transplantation. A rat orthotopic limb transplantation model was used to evaluate the effects of acute rejection on nerve regeneration and motor recovery. Continuous administration of FK506 (full suppression), administration of FK506 for the first 8 of 12 weeks (late rejection), or delayed administration of FK506/dexamethasone following noticeable rejection (early rejection) was used to preclude or induce rejection following limb transplantation. Twelve weeks postoperatively, nerve regeneration was assessed by means of histomorphometric analysis of explanted sciatic nerve, and motor recovery was assessed by means of evoked muscle force measurement in extensor digitorum longus muscle. A single episode of acute rejection that occurs immediately or late after reconstruction does not significantly alter the number of regenerating axonal fibers. Acute rejection occurring late after reconstruction adversely affects extensor digitorum longus muscle function in composite tissue allografts. Collected data reinforce that adequate immunosuppressant administration in cases of allogeneic limb transplantation ensures levels of nerve regeneration and motor functional recovery equivalent to that of syngeneic transplants. Prompt rescue following acute rejection was further demonstrated not to significantly affect nerve regeneration and functional recovery postoperatively. However, instances of acute rejection that occur late after reconstruction affect graft function. In total, the present study begins to characterize the effect of immunosuppression regimens on nerve regeneration and motor recovery in the setting of composite tissue allografts.

Targeted Disruption of Calcium/NFAT Signaling Reveals a Left-Right Determination Disorder in the Pharyngeal Arch Artery

An immunosuppressant was injected into pregnant mice in order to investigate whether the immune response is involved in differentiation during embryonic development. Injection of 3 mg/kg of FK506, an inhibitor of calcineurin, early in the organogenesis period increased the penetrance of right aortic arch formation by 32% compared with saline injection. Immunosuppressants such as FTY720 and rapamycin did not affect left/right (L/R) determination. FK506 is known to work by restricting NFAT (nuclear factor activated T-cell) dephosphorylation. An L/R determination disorder in cardiac outflows appeared when an NFATc4 siRNA was directly injected into the amniotic fluid. As for the mechanism, Pitx2, which is normally expressed on the left-hand side, was found to be expressed also on the right-hand side. Furthermore, it turned out that administration of FK506 also prevented the dephosphorylation of NSFL1 cofactor p47. When an siRNA targeting p47 was introduced into the amniotic fluid of FK506-treated fetuses, both of the dorsal arteries—which should normally become one—remained. These findings indicate that the mother's immune system contributes not only to self defense, but also to remodeling processes in fetal morphogenesis.

Effect of FK506 in reducing scar formation by inducing fibroblast apoptosis after sciatic nerve injury in rats

We previously demonstrated that FK506, a generally applied immunosuppressant in organ transplantation, could promote peripheral nerve regeneration through reducing scar formation. However, little is known about how FK506 reduces scar formation. Herein we investigated the influence of FK506 on fibroblast proliferation and its correlation with scar formation after sciatic nerve injury in rats, and further explored the effect of FK506 on fibroblast proliferation and apoptosis in vitro. Masson staining and immunohistochemistry revealed that scar area and fibroblast number in the nerve anastomosis of sciatic nerve-injured rats were significantly reduced after FK506 administration. The scar area had a significant positive correlation with the fibroblast number, as detected by linear correlation analysis. CCK-8 assay and flow cytometry indicated that FK506 also inhibited proliferation and induced apoptosis of fibroblasts in vitro. It was primarily phosphorylation of JNK and ERK that were activated during the apoptosis of fibroblast. Pretreatment of cells with JNK inhibitor, SP600125, or ERK inhibitor, PD98059, could inhibit FK506-induced fibroblast apoptosis, respectively. Moreover, simultaneous application of both inhibitors had additive roles in cell protection from apoptosis. These results suggest that FK506-induced fibroblast apoptosis contributes to the suppression of fibroblast proliferation and then results in the reduction of scar formation in sciatic nerve-injured rat, and that JNK and ERK are involved in FK506-induced fibroblast apoptosis.

Involvement of intestinal intraepithelial lymphocytes in turnover of intestinal epithelial cells: Morphological and functional alterations due to daily administration of FK506

To elucidate the interaction of intestinal intraepithelial lymphocytes (IELs) with intestinal epithelial cells (IECs), we investigated alterations of IECs by activating or inactivating IELs. The stimulation of IELs with anti-mouse CD3 monoclonal antibody induced massive apoptosis of IECs. Changes in IECs and IELs from mice that received daily administration of FK506 for 14days were investigated. IELs, particularly TCR-γδ+ IELs, were reduced in cell number, and a decrease of cytotoxic activity was observed. Under this condition, loss of apoptotic cells at the tips of villi and delayed turnover of IECs were detected. The expressions of alkaline phosphatase and CD98 amino acid transporters on IECs were decreased. Furthermore, abnormal skeletal organization of villi and weakened binding of IECs to the basement membrane were shown. These results suggest that inactivated IECs, which should be led to apoptosis, remained. It was strongly suggested that IELs participated in IEC turnover.

SAHA, an HDAC inhibitor, synergizes with tacrolimus to prevent murine cardiac allograft rejection

Suberoylanilide hydroxamic acid (SAHA), as a histone deacetylase (HDAC) inhibitor (HDACi), was recently found to exhibit an immunosuppressive effect. However, whether SAHA can synergize with calcineurin inhibitors (CNIs) to inhibit allograft rejection and its underlying mechanism remain elusive. In this study, we demonstrated the synergistic effects of SAHA and non-therapeutic dose of tacrolimus (FK506) in prolonging the allograft survival in a murine cardiac transplant model. Concomitant intragraft examination revealed that allografts from SAHA-treated recipients showed significantly lower levels of IL-17 expression, and no discernable difference for IL-17 expressions was detected between SAHA- and SAHA/FK506-treated allograft as compared with allografts from FK506-treated animals. In contrast, administration of FK506 significantly suppressed interferon (IFN)-γ but increased IL-10 expression as compared with that of SAHA-treated animals, and this effect was independent of SAHA. Interestingly, SAHA synergizes with FK506 to promote Foxp3 and CTLA4 expression. In vitro, SAHA reduced the proportion of Th17 cells in isolated CD4⁺ T-cell population and decreased expressions of IL-17A, IL-17F, STAT3 and RORγt in these cells. Moreover, SAHA enhances suppressive function of regulatory T (Treg) cells by upregulating the expression of CTLA-4 without affecting T effector cell proliferation, and increased the proportion of Treg by selectively promoting apoptosis of T effector cells. Therefore, SAHA, a HDACi, may be a promising immunosuppressive agent with potential benefit in conjunction with CNI drugs.

Inhibition of calcineurin in the prefrontal cortex induced depressive-like behavior through mTOR signaling pathway

Although it has been recognized that inhibition of calcineurin induced depressive-like behavior, the underlying neural mediators have not yet been identified. Mammalian target of rapamycin (mTOR), a serine/threonine protein kinase that regulates protein synthesis in synapses, has been demonstrated to be involved in the rapid antidepressant effects of ketamine. To investigate a potential role of mTOR signaling pathway which interferes with depressive-like behavior induced by calcineurin blockade and to determine the neurobiological mechanisms underlying mood-related disorders. Calcineurin inhibitor cyclosporine A (CsA) and tacrolimus (FK506) were microinjected into the medial prefrontal cortex (mPFC) in rats, and the depressive-like behavior was measured in sucrose preference test and forced swim test. Additionally, mTOR activity was tested by the levels of phosphorylation of p70s6 kinase (p70s6k) and 40S ribosomal protein S6 (rps6). Chronic microinjection of CsA or FK506 into mPFC increased depressive-like behaviors and decreased mTOR activity, but acute CsA or FK506 had no effects on both behavioral phenotype and mTOR activity. Furthermore, activation of mTOR by NMDA reversed the depressive-like behavior induced by chronic CsA or FK506 administration. Moreover, inhibition of mTOR by rapamycin reversed the antidepressant effects of ketamine. Finally, traditional antidepressant venlafaxine prevented the depressive-like performance induced by chronic CsA or FK506 treatment. These findings indicate that calcineurin-inhibition-induced depressive-like behavior is mediated by blockade of the mTOR signaling pathway and raise the possibility that stimulation of specific brain mTOR may be sufficient to decrease risk of affective disorders in patients treated with calcineurin inhibitor.

Geldanamycin accelerated peripheral nerve regeneration in comparison to FK-506 in vivo

FK-506 accelerates nerve regeneration and improves functional recovery in vivo; its immunosuppressive properties, however, limit its clinical utility. Geldanamycin (GA), a non-immunosuppressive agent, shares a common binding target (heat shock protein 90) with FK-506 and may accelerate nerve regeneration through a similar mechanism. GA has been shown to augment neurite outgrowth in vitro but has not been tested in vivo. The current study investigated the effect of GA on the rate of axonal regeneration and functional recovery following peripheral nerve injury. In the first experiment, Thy1-GFP transgenic rats underwent serial transmuscular imaging to quantify the rate of axonal regeneration following saphenous nerve crush injury. In subsequent experiments, Lewis rats underwent tibial nerve crush or transection-and-repair injuries and were assessed for functional recovery by walking track analysis. All animals were randomized to receive daily administration of FK-506 (2mg/kg), GA (0.2mg/kg), or a control vehicle (dimethyl sulfoxide, 1mL/kg) starting 3days prior to injury. Both GA and FK-506 significantly increased the rate of axonal regeneration following crush injury in Thy1-GFP rats. In Lewis rats undergoing tibial nerve crush injury, earlier functional recovery occurred at day 5 and day 6 in animals treated with FK-506 and GA respectively, vs. day 13 for controls. Over a truncated 21-day timeframe, Lewis rats undergoing tibial nerve transection-and-repair injury and treated with FK-506 regained function at day 16, whereas those treated with GA or the control vehicle did not regain normal function. GA-treated animals, however, did exhibit significant functional improvement vs. controls. The current study demonstrated that GA accelerates axonal regeneration and enhances functional recovery in vivo. Its ability to increase the rate at which peripheral axons regenerate is comparable to that of FK-506. GA, however, did not match the performance of FK-506 in injury models where Wallerian degeneration (WD) is ongoing in the distal stump. This provides evidence that FK-506 accelerates axonal regeneration through two parallel mechanisms: the first being its well-established effect on neurons; the second is likely a newly described, as-yet poorly defined mechanism that affects WD. Finally, given the decrease in observed toxicity with GA administration, it might be a suitable non-immunosuppressive alternative to FK-506 for accelerating peripheral nerve regeneration in cases of clinical nerve injury.

Efficacy of Short-Term FK506 Administration on Accelerating Nerve Regeneration

Background. The slow rate of nerve regeneration following injury can cause extended muscle denervation, leading to irreversible muscle atrophy, fibrosis, and destruction of motor endplates. The immunosuppressant FK506 (tacrolimus) has been shown to accelerate the rate of nerve regeneration and functional recovery. However, the toxic and immunosuppressive properties of FK506 make it undesirable for long-term use. Objective. To take advantage of the regeneration-enhancing effects of FK506 but avoid the potential adverse effects of long-term administration, the current study evaluates and quantifies the efficacy of short-term FK506 treatment in rat models. Methods. Clinically relevant transection and graft models were evaluated, and walking track analysis (WTA) was used to evaluate functional recovery. FK506 was administered for 5 and 10 days post transection injury and 10 and 20 days post graft injury. Both groups involving a short course were compared with the continuous administration group. Results. In the transection model, FK506 was administered for 5 and 10 days postoperatively. WTA demonstrated that 10 days of FK506 administration was sufficient to reduce functional recovery time by 29% compared with negative controls. In the graft model, FK506 was administered for 10 and 20 days postoperatively. Short treatment courses of 10 and 20 days reduced recovery time by 15% and 21%, respectively, compared with negative controls. Analysis of blood–nerve barrier (BNB) integrity demonstrated that FK506 facilitated early reconstitution of the BNB. Conclusions. The results of this study indicate that short-term FK506 delivery following nerve injury imparts a significant therapeutic effect.

Traumatic Brain Injury Causes an FK506-Sensitive Loss and an Overgrowth of Dendritic Spines in Rat Forebrain

Traumatic brain injury (TBI) causes both an acute loss of tissue and a progressive injury through reactive processes such as excitotoxicity and inflammation. These processes may worsen neural dysfunction by altering neuronal circuitry beyond the focally-damaged tissue. One means of circuit alteration may involve dendritic spines, micron-sized protuberances of dendritic membrane that support most of the excitatory synapses in the brain. This study used a modified Golgi-Cox technique to track changes in spine density on the proximal dendrites of principal cells in rat forebrain regions. Spine density was assessed at 1 h, 24 h, and 1 week after a lateral fluid percussion TBI of moderate severity. At 1 h after TBI, no changes in spine density were observed in any of the brain regions examined. By 24 h after TBI, however, spine density had decreased in ipsilateral neocortex in layer II and III and dorsal dentate gyrus (dDG). This apparent loss of spines was prevented by a single, post-injury administration of the calcineurin inhibitor FK506. These results, together with those of a companion study, indicate an FK506-sensitive mechanism of dendritic spine loss in the TBI model. Furthermore, by 1 week after TBI, spine density had increased substantially above control levels, bilaterally in CA1 and CA3 and ipsilaterally in dDG. The apparent overgrowth of spines in CA1 is of particular interest, as it may explain previous reports of abnormal and potentially epileptogenic activity in this brain region.

Combination therapy with bone marrow stromal cells and FK506 enhanced amelioration of ischemic brain damage in rats

AimsTransplantation of bone marrow stromal cells (MSCs) has been shown to ameliorate ischemic brain injury in animals. In the present study, we investigated whether the transplantation of MSCs combined with FK506, a clinically used immunosuppressant, enhanced neuroprotective effects in rat experimental stroke. Main methodsMale Sprague–Dawley rats underwent transient 90min middle cerebral artery occlusion (MCAO). Two or 6h after ischemia onset, the rats were randomly assigned to receive intravenous administration of MSCs plus FK506, MSCs alone, FK506 alone, or vehicle. Infarct volume, and neurological and immunohistological assessments were performed to examine the effects of these therapies. Key findingsIn 2-hour post-ischemia treatment groups, significant improvement of infarct volume and neurological scores were observed 1day after combination therapy compared with monotherapy, and this neuroprotection continued for 7days. Combination therapy significantly reduced the number of TUNEL-positive apoptotic cells, increased Bcl-2 expression, decreased Bax expression, and suppressed neutrophil infiltration and microglia/macrophage activation compared to monotherapy. In 6-hour post-ischemia treatment groups, a significant reduction of infarct volume, edema index, and neurological score was observed only in the combination therapy group. Moreover, the number of engrafted MSCs on day 7 with combination therapy was significantly higher than with MSCs alone. SignificanceCombination therapy using FK506 enhanced the anti-apoptotic and anti-inflammatory effects of MSCs and increased the survival of transplanted cells, leading to expansion of the therapeutic time window for MSCs.

FKBP12 Is a Critical Regulator of the Heart Rhythm and the Cardiac Voltage-Gated Sodium Current in Mice

FK506 binding protein (FKBP)12 is a known cis-trans peptidyl prolyl isomerase and highly expressed in the heart. Its role in regulating postnatal cardiac function remains largely unknown. We generated FKBP12 overexpressing transgenic (αMyHC-FKBP12) mice and cardiomyocyte-restricted FKBP12 conditional knockout (FKBP12(f/f)/αMyHC-Cre) mice and analyzed their cardiac electrophysiology in vivo and in vitro. A high incidence (38%) of sudden death was found in αMyHC-FKBP12 mice. Surface and ambulatory ECGs documented cardiac conduction defects, which were further confirmed by electric measurements and optical mapping in Langendorff-perfused hearts. αMyHC-FKBP12 hearts had slower action potential upstrokes and longer action potential durations. Whole-cell patch-clamp analyses demonstrated an ≈ 80% reduction in peak density of the tetrodotoxin-resistant, voltage-gated sodium current I(Na) in αMyHC-FKBP12 ventricular cardiomyocytes, a slower recovery of I(Na) from inactivation, shifts of steady-state activation and inactivation curves of I(Na) to more depolarized potentials, and augmentation of late I(Na), suggesting that the arrhythmogenic phenotype of αMyHC-FKBP12 mice is attributable to abnormal I(Na). Ventricular cardiomyocytes isolated from FKBP12(f/f)/αMyHC-Cre hearts showed faster action potential upstrokes and a more than 2-fold increase in peak I(Na) density. Dialysis of exogenous recombinant FKBP12 protein into FKBP12-deficient cardiomyocytes promptly recapitulated alterations in I(Na) seen in αMyHC-FKBP12 myocytes. FKBP12 is a critical regulator of I(Na) and is important for cardiac arrhythmogenic physiology. FKPB12-mediated dysregulation of I(Na) may underlie clinical arrhythmias associated with FK506 administration.

Effect of a calcineurin inhibitor tacrolimus (FK506) treatment on meiotic chromosomes in testes, epididymal spermatozoa and fertility in Swiss Albino male mice

Tacrolimus hydrate (FK506), isolated from Streptomyces tsukubaensis, is an immensely used immunosuppressive agent. It was evaluated for its effects on meiotic chromosomes in testes, epididymal spermatozoa and fertility in male mice. The study under different parameters constituted sub acute (seven days) administration (gavage) of FK506 at doses of 4, 8 and 16 mg/kg/day body weight. The results obtained in present study revealed that, FK506 significantly induced spermatozoa abnormalities, lowered fertility and increased embryonic loss. The observed changes related to spermatogenic dysfunction reflected statistically significant increased aberrations in the meiotic chromosomes. These aberrations seemed to be induced by the inhibitory effect of the drug on the signal transduction pathways in the cells and interference in the transcription processes and proliferation of cells. Further studies are warranted to evaluate the safe dose and duration of therapy to determine the exact mode of action of FK506 induced germ cell toxicity. Key words : Tacrolimus (FK506), sperm quality, germ cell toxicity, embryonic loss.