Towards novel treatments for adrenal diseases: Cell- and gene therapy-based approaches.

Adrenal insufficiency, the inability to produce adequate levels of corticosteroids, is a multi-causal disease that requires lifelong daily hormone replacement. Nevertheless, this cannot replace the physiological demand for steroids which are secreted following a circadian rhythm and vary in periods of stress; the consequences of under- or over-replacement include adrenal crisis and metabolic disturbances, respectively. Although clinical research has focused on enhancing the effectiveness/reducing side effects of current treatment modalities, only small improvements are deemed possible; thus, alternative solutions are urgently needed. Gene and cell therapy strategies have opened new possibilities for the cure of many diseases in a way that has never been possible before and could offer a viable option for the cure of adrenal diseases. The current state of cell- and gene-based approaches to restore adrenocortical function is discussed in this review.

Extension of Survival in Bilaterally Adrenalectomized Mice by Implantation of SF-1/Ad4BP-Induced Steroidogenic Cells.

Mesenchymal stroma/stem cells (MSCs) exist in adult tissues, such as adipose tissue and bone marrow, and differentiate into cells of multiple lineages. In previous studies, we found that MSCs differentiate into steroidogenic cells by forced expression of steroidogenic factor 1 (SF-1)/adrenal 4 binding protein (Ad4BP), the master regulator of steroidogenesis and differentiation of pituitary gonadotrophs, adrenal glands, and gonads. In this study, SF-1/Ad4BP-induced steroidogenic cells derived from mouse adipose tissue-derived MSCs (ADSCs) were implanted under the kidney capsule of bilateral adrenalectomized (bAdx) mice. bAdx mice did not survive after 7 days. However, 4 of 9 bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, 1 of 10 bAdx mice transplanted with control ADSCs, and bAdx mice transplanted with an adrenal gland survived for 30 days. Plasma corticosterone levels in bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells and control ADSCs were 5.41 ±â€…2.26 ng/mL (mean ±â€…SEM) and undetectable at 7 days after implantation, respectively. After removal of the kidney bearing the graft from the surviving mice at 30 days after implantation, plasma corticosterone was not detected in any of the samples. Immunohistochemical staining revealed SF-1/Ad4BP-positive cells under the capsule of the kidney. Although we performed an adrenocorticotropin (ACTH) loading test on bAdx mice implanted with SF-1/Ad4BP-induced steroidogenic cells, ACTH responsiveness was not observed. Implantation of steroidogenic cells derived from ADSCs into bAdx mice increased the basal plasma corticosterone level and extended the survival of bAdx mice, suggesting the capability of restoring steroidogenic cells by cell transplantation therapy for adrenal insufficiency.

Autologous Adrenal Transplantation for the Treatment of Refractory Cushing's Disease.

OBJECTIVE: To report our management of bilateral adrenalectomy with autologous adrenal gland transplantation for persistent Cushing's disease, and to discuss the feasibility of autologous adrenal transplantation for the treatment of refractory Cushing's disease. MATERIAL AND METHODS: A retrospective analysis was performed in 4 patients (3 females, aged 14-36 years) who underwent autologous adrenal transplantation for persistent Cushing's disease after endonasal transsphenoidal resection of a pituitary tumor. The procedure was performed by implanting a vascularized adrenal graft into the left iliac fossa with direct and indirect anastomoses. Postoperative follow-up was performed in 1, 1.5, 8, and 10 years, and an over 8-year long-term follow-up was reached in 2 out of the 4 cases. Hormone replacement dosage was guided by clinical symptoms and endocrine results including serum cortisol (F), 24 h urine-free cortisol, and adrenocorticotrophic hormone levels. RESULTS: All 4 patients with symptomatic Cushing's disease experienced resolution of symptoms after autotransplantation without Nelson Syndrome. Functional autografts were confirmed through clinical evaluation and endocrine results. One year after transplantation, adrenal function and hormone replacement dosage remained stable without adrenal hyperplasia. After long-term follow-up, dosages of hormone replacement were reduced in all patients. CONCLUSIONS: In this series of 4 patients, we demonstrate the long-term efficacy of bilateral adrenalectomy with autologous adrenal transplantation and propose this procedure as a viable treatment option for refractory Cushing's disease.

Autogenous transplants of adrenal fragments in an animal model.

Introduction Adrenal insufficiency is a typical complication after surgical treatment of adrenal tumors, especially after the removal of both adrenal glands. Human beings are not able to survive without adrenal glands and without proper hormonal substitution. Autotransplantation of a fragment of the adrenal gland may prevent this complication. This can be done by transplanting the entire adrenal glands or its fragment, such as the adrenal cortex cells. In the case of adrenal tumors, the entire adrenal gland can not be transplanted. However, it is possible to transplant cells from the tumor-free part. Succesful adrenal autografts may result in a new treatment of adrenal insufficiency. MATERIALS AND METHODS: Autograft transplantation was performed on 3 groups of Sprague Dawley rats. In the first group, physiological corticosterone concentrations were determined. These animals were not operated. In the second group, both adrenal glands were removed. Corticosterone concentrations were determined after bilateral adrenalectomy. The third group was divided into two parts. In the first subgroup, bilateral adrenalectomy was performed simultaneosly with adrenal transplant into the omentum. In the second subgroup, right adrenalectomy was performed simultaneosly with and adrenal transplant into the omentum followed a month later by left adrenalectomy. During the experiment, corticosterone concentrations were measured at 4 time points. RESULTS: The statistical difference between corticosterone concentrations in rats after two timed adrenalectomies and rats after bilateral adrenalectomy was statistically different, but these results were far from physiological concentrations.

Involvement of DHH and GLI1 in adrenocortical autograft regeneration in rats.

Bilateral adrenalectomy forces the patient to undergo glucocorticoid replacement therapy and bear a lifetime risk of adrenal crisis. Adrenal autotransplantation is considered useful to avoid adrenal crisis and glucocorticoid replacement therapy. However, the basic process of regeneration in adrenal autografts is poorly understood. Here, we investigated the essential regeneration factors in rat adrenocortical autografts, with a focus on the factors involved in adrenal development and steroidogenesis, such as Hh signalling. A remarkable renewal in cell proliferation and increase in Cyp11b1, which encodes 11-beta-hydroxylase, occurred in adrenocortical autografts from 2-3 weeks after autotransplantation. Serum corticosterone and adrenocorticotropic hormone levels were almost recovered to sham level at 4 weeks after autotransplantation. The adrenocortical autografts showed increased Dhh expression at 3 weeks after autotransplantation, but not Shh, which is the only Hh family member to have been reported to be expressed in the adrenal gland. Increased Gli1 expression was also found in the regenerated capsule at 3 weeks after autotransplantation. Dhh and Gli1 might function in concert to regenerate adrenocortical autografts. This is the first report to clearly show Dhh expression and its elevation in the adrenal gland.

PreImplantation Factor (PIF*) Regulates Stress-Induced Adrenal Steroidogenesis and Anti-Inflammatory Cytokines: Potential Application for Bioartificial Adrenal Transplant.

The main treatment algorithm for adrenal insufficiency is hormonal replacement, however, inadequate hormone substitution often leads to severe side effects. Adrenal cell transplantation could be a more effective alternative but would require life-long immune suppressive therapy. PreImplantation Factor (PIF) is an endogenous peptide secreted by viable human embryos that leads to maternal tolerance without immunosuppression. PIF could be effective for xenogeneic cell transplantation such as of bovine adrenocortical cells (BAC), which are used for bioartificial adrenal gland development that may more effectively restore complex adrenal functions. We report here that PIF exerts a dual regulatory effect on BAC by targeting mostly hyper-activated cells to specifically reduce adrenocorticotropic hormone (ACTH)-stimulated cortisol secretion. Reverse transcription real time PCR analysis revealed that PIF modulates the expression of two genes in the cortisol synthesis pathway, Steroidogenic Factor 1 (SF1), an activator of steroidogenesis, and the downstream steroidogenic enzyme Cytochrome P450 17A1 (CYP17A1). PIF increased basal expression of SF1 and CYP17A1 regardless of the activation level of the adrenocortical cells. In contrast, following ACTH stimulation, PIF reduced SF1 expression and induced expression of the immune suppressing anti-inflammatory cytokine IL10 only in the hyper-activated cells, suggesting both a protective and immune tolerant function. In conclusion, PIF regulates stress-induced adrenal steroidogenesis and immune tolerance in BAC, supporting a potential clinical application to reduce rejection by the host's immune response following xenotransplantation.

Spinal cord injury-induced immunodeficiency is mediated by a sympathetic-neuroendocrine adrenal reflex.

Acute spinal cord injury (SCI) causes systemic immunosuppression and life-threatening infections, thought to result from noradrenergic overactivation and excess glucocorticoid release via hypothalamus-pituitary-adrenal axis stimulation. Instead of consecutive hypothalamus-pituitary-adrenal axis activation, we report that acute SCI in mice induced suppression of serum norepinephrine and concomitant increase in cortisol, despite suppressed adrenocorticotropic hormone, indicating primary (adrenal) hypercortisolism. This neurogenic effect was more pronounced after high-thoracic level (Th1) SCI disconnecting adrenal gland innervation, compared with low-thoracic level (Th9) SCI. Prophylactic adrenalectomy completely prevented SCI-induced glucocorticoid excess and lymphocyte depletion but did not prevent pneumonia. When adrenalectomized mice were transplanted with denervated adrenal glands to restore physiologic glucocorticoid levels, the animals were completely protected from pneumonia. These findings identify a maladaptive sympathetic-neuroendocrine adrenal reflex mediating immunosuppression after SCI, implying that therapeutic normalization of the glucocorticoid and catecholamine imbalance in SCI patients could be a strategy to prevent detrimental infections.

The first simultaneous kidney-adrenal gland-pancreas transplantation: outcome at 1 year.

Adrenal insufficiency is a rare but life-threatening disease. Replacement therapy sometimes fails to prevent an acute adrenal crisis and most often does not lead to restoration of well-being. We report here the 1-year outcome of the first simultaneous kidney-adrenal gland-pancreas transplantation in a 33-year-old patient with type 1 diabetes and concomitant autoimmune adrenal insufficiency. En bloc left adrenal gland and kidney grafts were anastomosed on the left iliac vessels in normal vascular conditions and the pancreas graft was anastomosed on the right iliac vessels. The immunosuppressive regimen was not modified by the addition of the adrenal gland. We observed no additional morbidity due to the adrenal gland transplantation, as there were no surgical complications. One-year kidney and pancreas graft functions were satisfactory (estimated glomerular filtration rate: 55 mL/min/1.73 m(2) and HbA1c: 4.8%). The adrenal graft functioned well at 12 months with a normalization of cortisol and aldosterone baseline levels. Functional imaging at 3 months showed good uptake of [(123) I]-metaiodobenzylguanidine by the adrenal graft. Transplantation of the adrenal gland en bloc with the left kidney appears to be a good therapeutic option in patients with adrenal insufficiency awaiting kidney or kidney-pancreas transplantation.

Adrenal-specific scavenger receptor BI deficiency induces glucocorticoid insufficiency and lowers plasma very-low-density and low-density lipoprotein levels in mice.

OBJECTIVE: We determined the physiological consequences of adrenocortical-specific deletion of scavenger receptor BI (SR-BI) function in C57BL/6 wild-type mice. METHODS AND RESULTS: One adrenal from 10-day-old SR-BI knockout (KO) mice or wild-type controls was transplanted under the renal capsule of adrenalectomized C57BL/6 recipient mice. The fasting plasma corticosterone level increased over time in transplanted mice. Corticosterone values in SR-BI KO transplanted mice remained ≈50% lower (P<0.001) as compared with wild-type transplanted mice, which coincided with adrenocortical lipid depletion. A 6.5-fold higher (P<0.01) plasma adrenocorticotropic hormone level was present in SR-BI KO transplanted mice reminiscent of primary glucocorticoid insufficiency. On feeding with cholic acid-containing high cholesterol/high fat diet, SR-BI KO transplanted mice exhibited a 26% (P<0.05) reduction in their liver triglyceride level. Hepatic myosin regulatory light chain interacting protein/inducible degrader of the low-density lipoprotein receptor mRNA expression was 48% (P<0.01) decreased in adrenal-specific SR-BI KO mice, which was paralleled by a marked decrease (-46%; P<0.01) in proatherogenic very-low-density and low-density lipoprotein levels. CONCLUSIONS: Adrenal-specific disruption of SR-BI function induces glucocorticoid insufficiency and lowers plasma very-low-density and low-density lipoprotein levels in atherogenic diet-fed C57BL/6 mice. These findings further highlight the interaction between adrenal high-density lipoprotein-cholesterol uptake by SR-BI, adrenal steroidogenesis, and the regulation of hepatic lipid metabolism.

Outcome of adrenal tissue fragments allotransplantation: the impact of cryopreservation.

Cryopreservation is thought to have the potential to preserve tissue for transplantation. In addition, it can also be used for decreasing tissue immunogenicity, which might be important for prolonging allograft survival. In the present study we examined the impact of cryopreservation at various cooling rates on the outcome of allotransplantation of murine adrenal tissue fragments (ATFr). ATFr were cryopreserved with a cooling rate at 1; 10; 40 and more than 100 °C/min. After thawing it was found that the number of the cells expressing markers of dendritic cells (CD11c) and macrophages (CD11b) in the suspension obtained from ATFr decreased with increasing cooling rate. After allotransplantation the survival rates of adrenalectomized mice and the blood serum levels of corticosterone were higher in recipients of cryopreserved ATFr. By immunohistochemistry, cryopreserved allografts displayed a decreased infiltration by CD4+ and CD8+ T-lymphocytes as compared to fresh grafts. These findings suggest that cryopreserved allografts cause a less severe rejection by decreasing graft immunogenicity.

Adrenalectomy stimulates the formation of initial atherosclerotic lesions: reversal by adrenal transplantation.

Long-term changes in the secretion of immunosuppressive adrenal-derived glucocorticoid hormones influence cardiovascular disease risk. Here we determined the consequences of changes in adrenal steroid metabolism for the development of atherosclerotic lesions in mice. Atherosclerosis-susceptible low-density-lipoprotein (LDL) receptor knockout mice were subjected to adrenalectomy (ADX) or a control (SHAM) operation and subsequently fed an atherogenic diet for 4 weeks. Atherogenic diet feeding raised plasma corticosterone levels in SHAM mice, but not adrenalectomized mice, resulting in an 83% lower (P<0.01) corticosterone level in adrenalectomized mice. Adrenalectomy was associated with a respectively 22% and 29% lower plasma level of cholesterol and triglycerides. In contrast, white blood cell counts were increased 2-fold (P<0.01) in adrenalectomized mice, which could be attributed to a significant 2.1- to 2.6-fold rise in lymphocyte (P<0.05) and monocyte (P<0.05) numbers. Probably as a result of the enhanced systemic inflammatory status, adrenalectomy was associated with a higher susceptibility for diet-induced atherosclerosis (321±18×10(3) µm(2) for ADX vs 240±31×10(3) µm(2) for SHAM; P<0.05) not withstanding the lowered cholesterol levels. Restoring adrenocortical steroid secretion - but not adrenal medulla function - and the associated downstream glucocorticoid receptor signaling in adrenalectomized mice through adrenal transplantation induced a reversal of the adrenalectomy-associated rise in white blood cell numbers, plasma monocyte chemoattractant protein 1 (MCP-1) levels, and atherosclerotic lesion development (lesion size in transplanted mice: 258±34×10(3) µm(2); P<0.05 vs ADX). In conclusion, our studies show that adrenal-derived steroids protect against the development of initial atherosclerotic lesions in LDL receptor knockout mice.

Effects on plasma corticosterone levels and brain serotonin from interference with methamphetamine-induced corticosterone release in neonatal rats.

Methamphetamine (MA) induces multiple effects in rats including alterations to corticosterone (CORT) and adrenocorticotropic hormone (ACTH). This effect is age dependent showing a U-shaped function similar to that of other stressors during the stress hyporesponsive period. Neonatal MA treatment leads to adult learning and memory impairments, but whether these are related to MA-induced CORT release is unknown. Here in, four methods were tested in neonatal rats previously established in adult rats for inhibiting stress-induced CORT release: inhibiting synthesis (metyrapone (MET) or ketoconazole (KTZ)) or surgically by adrenalectomy or adrenal autotransplantation (ADXA). Pretreatment on postnatal day 11 with MET or KTZ prior to four doses of 10 mg/kg of MA initially suppressed MA-induced increases in plasma CORT, but 24 h later, even with additional inhibitor treatment, a large CORT increase was seen which exceeded that of MA alone. Adrenalectomy blocked MA-induced increases in CORT but caused a secondary effect on brain serotonin (5-HT) and dopamine (DA), causing greater reductions than those caused by MA alone. ADXA inhibited MA-induced CORT release without causing a 24-h CORT increase and did not produce additional effects on brain 5-HT or DA. Neonatal ADXA is a new model for developmental drug or stress experiments designed to test the role of CORT in mediating early effects on later outcomes.

Effects of inhibiting neonatal methamphetamine-induced corticosterone release in rats by adrenal autotransplantation on later learning, memory, and plasma corticosterone levels.

RATIONALE: Neonatal rat methamphetamine (MA) exposure has been shown to cause long-term behavioral impairments similar to some of those observed following neonatal stress. The mechanism by which MA induces impairments is unknown but may be related to early increases in corticosterone release. We previously developed a method to attenuate MA-induced corticosterone release using adrenal autotransplantation (ADXA) in neonatal rats. This exposure period corresponds to the second-half of human pregnancy. OBJECTIVE: To determine whether inhibition of neonatal MA-induced increases in corticosterone attenuates the long-term behavioral deficits associated with early MA treatment. RESULTS: ADXA successfully attenuated MA-induced plasma corticosterone increases by approximately 50% during treatment (P11-20) but did not attenuate the long-term behavioral effects of MA treatment. MA-treated rats, regardless of surgery, showed increased errors and latencies in the Cincinnati water maze test of egocentric learning and increased latency, path length, and cumulative distance in three phases of Morris water maze spatial learning and reference memory. MA-treated offspring were hypoactive, had subtle reductions in anxiety in the elevated zero maze but not in the light-dark test. ADXA had no effect on MA-induced long-term 5-HT reductions in the neostriatum or entorhinal cortex or on 5-HIAA reductions in the hippocampus. CONCLUSIONS: Fifty percent attenuation of neonatal MA-induced elevations in corticosterone does not alter the long-term egocentric or allocentric learning deficits or other behavioral effects of neonatal MA exposure. Because the ADXA effect was partial, the data cannot rule out the possibility that a more complete block of MA-induced corticosterone release might not prevent later cognitive deficits.

Neonatal methamphetamine-induced corticosterone release in rats is inhibited by adrenal autotransplantation without altering the effect of the drug on hippocampal serotonin.

Rat neonatal methamphetamine exposure results in corticosterone release and learning and memory impairments in later life; effects also observed after neonatal stress. Previous attempts to test the role of corticosterone release after methamphetamine using corticosterone inhibitors were unsuccessful and adrenalectomy caused reductions in hippocampal serotonin greater than those caused by methamphetamine alone. Here we tested whether adrenal autotransplantation could be used to attenuate methamphetamine-induced corticosterone release without also altering the effects of the drug on serotonin. Adrenal autotransplantation surgery occurred on postnatal day 9 followed by methamphetamine or saline treatment from postnatal day 11-20 (10mg/kg/dosex4/day). Plasma corticosterone and hippocampal serotonin and 5-hydroxyindoleacetic acid were determined 30min following the first treatment on each day between postnatal days 11-20. Adrenal autotransplantation attenuated neonatal methamphetamine-induced corticosterone release by approximately 70% initially, approximately 55% midway through treatment, and approximately 25% by the end of treatment. Methamphetamine reduced serotonin and 5-hydroxyindoleacetic acid in the hippocampus in the ADXA rats to the same degree as in SHAM rats. The data show that neonatal adrenal autotransplantation is an effective method for partially reducing treatment-induced corticosterone release while providing sufficient corticosterone to sustain normal growth and development. The method should be applicable to other models of developmental stress/corticosterone release.

A case of human intramuscular adrenal gland transplantation as a cure for chronic adrenal insufficiency.

Intramuscular endocrine gland transplantation has been well described as it pertains to parathyroid autotransplantation; however, transplantation of the adrenal gland is less well characterized. While adrenal autotransplantation in the setting of Cushing's disease has been described, intramuscular adrenal allotransplantation as a cure for adrenal insufficiency to our knowledge has not been previously carried out. Current treatment for adrenal insufficiency leaves patients without diurnal variation in cortisol release and susceptible to the detrimental effects of chronic hypercortisolism. We describe here the case of a 5-year-old girl with renal failure who had adrenal insufficiency following fulminant meningococcemia that led to requirements for both stress-dose steroid and mineralocorticoid replacement. Ten months after the onset of her disease, she received a simultaneous renal and adrenal gland transplant from her mother. The adrenal gland allograft was morselized into 1 mm(3) segments and implanted into three 2 cm pockets created in her rectus abdominis muscle. Three years after surgery, her allograft remains fully functional, responding well to adrenocorticotropin hormone stimulation and the patient does not require any steroid or mineral-corticoid supplementation. We believe this case represents the first description of successful functional intramuscular adrenal allograft transplantation with long-term follow up as a cure for adrenal insufficiency.

Aggressive behaviors in adult SF-1 knockout mice that are not exposed to gonadal steroids during development.

Sex hormones are a major factor responsible for the development of sex differences. Steroidogenic factor 1 (SF-1) is a key regulator of gonadal and adrenal development, and SF-1 knockout mice (SF-1 KO) are born without gonads and adrenal glands. Consequently, these mice are not exposed to gonadal sex steroids. SF-1 KO pups die shortly after birth due to adrenal deficiency. In the present study, SF-1 KO mice were rescued by neonatal corticosteroid injections followed by adrenal transplantations on day 7-8 postnatally. Control mice received corticosteroid injections and were gonadectomized prior to puberty. Mice were observed interacting with ovariectomized hormone primed females and gonad-intact males. In the absence of sex steroid replacement, adult SF-1 KO mice were significantly more aggressive than control mice in tests with stimulus females. After testosterone treatment, control males displayed significantly more aggression towards male intruders than control female mice, or male and female SF-1 KO mice, suggesting a developmental role of gonadal hormones in the expression of aggressive behavior and affirming SF-1 KO mice as a behavioral model to investigate affects of fetal gonad deficiency.

Bilateral subthalamic deep brain stimulation in a patient with Parkinson's disease who had previously undergone thalamotomy and autologous adrenal grafting in the caudate nucleus: case report.

OBJECTIVE: Subthalamic (Stn) deep brain stimulation (DBS) is a valid surgical therapy for the treatment of severe Parkinson's disease. In recent years, StnDBS has been proposed for patients who previously received other surgical treatments, such as thalamotomy and pallidotomy. Nonetheless, there is no consensus about the indications of DBS in patients who previously underwent surgery. To the best of our knowledge this is the first reported case of a patient treated with DBS after previous thalamotomy and adrenal grafting. CLINICAL PRESENTATION: A 62-year-old man with a long history (more than 30 yr) of Parkinson's disease received unilateral thalamotomy and autologous adrenal graft on two independent occasions. Thalamotomy led to a significant improvement, although limited to the control of contralateral tremor. The autologous adrenal graft was of no benefit. For the subsequent occurrence of L-dopa related dyskinesias and severe "off" periods, the patient was referred to our center for StnDBS. INTERVENTION: The patient underwent bilateral StnDBS, obtaining a satisfactory improvement of rigidity and bradykinesia on both sides. The 1-year follow-up evaluation showed a 46% improvement in the Unified Parkinson's Disease Rating Scale motor section, along with a noticeable reduction in antiparkinsonian therapy (81%). CONCLUSION: This case is consistent with previous reports from the literature, suggesting that StnDBS is feasible and safe, even in patients who previously received other surgical treatments for Parkinson's disease, such as thalamotomy or cell grafting.