Psoriasiform skin disease in transgenic pigs with high-copy ectopic expression of human integrins α2 and ß1.

Psoriasis is a complex human-specific disease characterized by perturbed keratinocyte proliferation and a pro-inflammatory environment in the skin. Porcine skin architecture and immunity are very similar to that in humans, rendering the pig a suitable animal model for studying the biology and treatment of psoriasis. Expression of integrins, which is normally confined to the basal layer of the epidermis, is maintained in suprabasal keratinocytes in psoriatic skin, modulating proliferation and differentiation as well as leukocyte infiltration. Here, we generated minipigs co-expressing integrins α2 and ß1 in suprabasal epidermal layers. Integrin-transgenic minipigs born into the project displayed skin phenotypes that correlated with the number of inserted transgenes. Molecular analyses were in good concordance with histological observations of psoriatic hallmarks, including hypogranulosis and T-lymphocyte infiltration. These findings mark the first creation of minipigs with a psoriasiform phenotype resembling human psoriasis and demonstrate that integrin signaling plays a key role in psoriasis pathology.

Leptin deficiency in mice counteracts imiquimod (IMQ)-induced psoriasis-like skin inflammation while leptin stimulation induces inflammation in human keratinocytes.

Leptin is an adipocyte-derived cytokine secreted mostly by adipose tissue. Serum leptin levels are elevated in obese individuals and correlate positively with body mass index (BMI). Interestingly, serum leptin levels are also elevated in patients with psoriasis and correlate positively with disease severity. Psoriasis is associated with obesity; patients with psoriasis have a higher incidence of obesity, and obese individuals have a higher risk of developing psoriasis. Additionally, obese patients with psoriasis experience a more severe degree of psoriasis. In this study, we hypothesised that leptin may link psoriasis and obesity and plays an aggravating role in psoriasis. To investigate leptin's role in psoriasis, we applied the widely accepted imiquimod (IMQ)-induced psoriasis-like skin inflammation mouse model on leptin-deficient (ob/ob) mice and evaluated psoriasis severity. Moreover, we stimulated human keratinocytes with leptin and investigated the effect on proliferation and expression of pro-inflammatory proteins. In ob/ob mice, clinical signs of erythema, infiltration and scales in dorsal skin and inflammation in ear skin, as measured by ear thickness, were attenuated and compared with wt mice. Moreover, IL-17A and IL-22 mRNA expression levels, as well as increased epidermal thickness, were significantly less induced. In vitro, the effect of leptin stimulation on human keratinocytes demonstrated increased proliferation and induced secretion of several pro-inflammatory proteins; two hallmarks of psoriasis. In conclusion, leptin deficiency attenuated IMQ-induced psoriasis-like skin inflammation in a mouse model, and leptin stimulation induced a pro-inflammatory phenotype in human keratinocytes, thus, supporting an aggravating role of leptin in psoriasis.

AZ17: a new bispecific drug targeting IL-6 and IL-23 with potential clinical use--improves psoriasis in a human xenograft transplantation model.

Targeting more than one molecule in multifactorial diseases involving several disease mediators may provide improved therapeutic efficacy. Psoriasis is a multifactorial disease in which interleukin (IL)-6 and IL-23 are important disease mediators because they facilitate development of Th17 cells; widely accepted to be associated with psoriasis. To meet the need for new therapeutics, we aimed to create a clinically relevant bispecific drug, by combining the inhibitory properties of anti-IL-6 and anti-IL-23 antibodies, exhibiting high affinity, high stability and the ability to be produced in high yield. The bispecific molecule AZ17 was created by combining high affinity binding domains originating from monoclonal antibodies targeting human IL-6 and IL-23. To allow for high and efficient production, AZ17 was assembled by site-specific bioconjugation from two individual single chain fragment variables that were synthesized separately in Escherichia coli. To improve stability and extend pharmacokinetics, a flexible poly-ethylene glycol molecule was used as linker. In preclinical psoriasis models, AZ17 reduced IL-23-induced ear inflammation and improved psoriasis in a xenograft transplantation model where psoriasis skin is transplanted onto immune-deficient mice. The data presented here suggest AZ17 to be a promising drug candidate in psoriasis and other inflammatory diseases associated with Th17 cell development.

Robust Lentiviral Gene Delivery But Limited Transduction Capacity of Commonly Used Adeno-Associated Viral Serotypes in Xenotransplanted Human Skin.

Skin is an easily accessible organ, and therapeutic gene transfer to skin remains an attractive alternative for the treatment of skin diseases. Although we have previously documented potent lentiviral gene delivery to human skin, vectors based on adeno-associated virus (AAV) rank among the most promising gene delivery tools for in vivo purposes. Thus, we compared the potential usefulness of various serotypes of recombinant AAV vectors and lentiviral vectors for gene transfer to human skin in a xenotransplanted mouse model. Vector constructs encoding firefly luciferase were packaged in AAV capsids of serotype 1, 2, 5, 6, 8, and 9 and separately administered by intradermal injection in human skin transplants. For all serotypes, live bioimaging demonstrated low levels of transgene expression in the human skin graft, and firefly luciferase expression was observed primarily in neighboring tissue outside of the graft. In contrast, gene delivery by intradermally injected lentiviral vectors was efficient and led to extensive and persistent firefly luciferase expression within the human skin graft only. The study demonstrates the limited capacity of single-stranded AAV vectors of six commonly used serotypes for gene delivery to human skin in vivo.

Resveratrol ameliorates imiquimod-induced psoriasis-like skin inflammation in mice.

BACKGROUND: The polyphenol resveratrol has anti-inflammatory effects in various cells, tissues, animals and human settings of low-grade inflammation. Psoriasis is a disease of both localized and systemic low-grade inflammation. The Sirtuin1 enzyme thought to mediate the effects of resveratrol is present in skin and resveratrol is known to down regulate NF-κB; an important contributor in the development of psoriasis. Consequently we investigated whether resveratrol has an effect on an Imiquimod induced psoriasis-like skin inflammation in mice and sought to identify candidate genes, pathways and interleukins mediating the effects. METHODS: The study consisted of three treatment groups: A control group, an Imiquimod group and an Imiquimod+resveratrol group. Psoriasis severity was assessed using elements of the Psoriasis Area Severity Index, skin thickness measurements, and histological examination. We performed an RNA microarray from lesional skin and afterwards Ingenuity pathway analysis to identify affected signalling pathways. Our microarray was compared to a previously deposited microarray to determine if gene changes were psoriasis-like, and to a human microarray to determine if findings could be relevant in a human setting. RESULTS: Imiquimod treatment induced a psoriasis-like skin inflammation. Resveratrol significantly diminished the severity of the psoriasis-like skin inflammation. The RNA microarray revealed a psoriasis-like gene expression-profile in the Imiquimod treated group, and highlighted several resveratrol dependent changes in relevant genes, such as increased expression of genes associated with retinoic acid stimulation and reduced expression of genes involved in IL-17 dependent pathways. Quantitative PCR confirmed a resveratrol dependent decrease in mRNA levels of IL-17A and IL-19; both central in developing psoriasis. CONCLUSIONS: Resveratrol ameliorates psoriasis, and changes expression of retinoic acid stimulated genes, IL-17 signalling pathways, IL-17A and IL-19 mRNA levels in a beneficial manner, which suggests resveratrol, might have a role in the treatment of psoriasis and should be explored further in a human setting.

Exploring Valrubicin's Effect on Propionibacterium Acnes-Induced Skin Inflammation in Vitro and in Vivo.

Acne is a common skin disease involving colonization with Propionibacterium acnes (P. acnes), hyperproliferation of the follicular epithelium and inflammatory events. Valrubicin is a second-generation anthracycline, non-toxic upon contact, and available in a topical formulation. Valrubicin's predecessor doxorubicin possesses antibacterial effects and previously we demonstrated that valrubicin inhibits keratinocyte proliferation and skin inflammation suggesting beneficial topical treatment of acne with valrubicin. This study aims to investigate valrubicin's possible use in acne treatment by testing valrubicin's antibacterial effects against P. acnes and P. acnes-induced skin inflammation in vitro and in vivo. Valrubicin was demonstrated not to possess antibacterial effects against P. acnes. Additionally, valrubicin was demonstrated not to reduce mRNA and protein expression levels of the inflammatory markers interleukin (IL)-1ß, IL-8, and tumor necrosis factor (TNF)-α in vitro in human keratinocytes co-cultured with P. acnes. Moreover, in vivo, valrubicin, applied both topically and intra-dermally, was not able to reduce signs of inflammation in mouse ears intra-dermally injected with P. acnes. Taken together, this study does not support beneficial antibacterial and anti inflammatory effects of topical valrubicin treatment of acne.

Debio 0932, a new oral Hsp90 inhibitor, alleviates psoriasis in a xenograft transplantation model.

Debio 0932 is a novel oral heat shock protein 90 (Hsp90) inhibitor developed for anti-cancer therapy. Surprising-ly, during the first clinical trial, one psoriasis patient experienced complete remission of his skin manifestation. However, a possible therapeutic utility of Hsp90 in psoriasis has not previously been reported. The objective of the present study was to explore the ability of Debio 0932 to alleviate psoriasis in a preclinical model. A psoriasis xenograft transplantation model was employed where skin from 5 psoriasis patients was transplanted onto immunodeficient mice (8 xenografts per donor). Debio 0932 was administered perorally daily for 3 weeks and resulted in significant clinical alleviation of psoriasis by day 11 and reduced epidermal thickness evaluated post-treatment. Alleviation of psoriasis in the psoriasis xenograft transplantation model, which may be due to Hsp90's involvement in signalling pathways that are up-regulated in psoriasis, substantiates a potential role of Debio 0932 in psoriasis treatment.

Valrubicin activates PKCa in keratinocytes: a conceivable mode of action in treating hyper-proliferative skin diseases.

BACKGROUND: Valrubicin is a semisynthetic anthracycline developed as an anti-cancer drug able to ameliorate psoriasis and non-melanoma skin cancer (NMSC) by topical application in animal models. Valrubicin decreases cell proliferation, and induces apoptosis; however its mode of action is still unknown. Valrubicin localizes in the cytoplasm and its valerate moiety resembles diacylglycerol, an activator of protein kinase C (PKC) α, which belongs to the PKC family of cytoplasmic serine/threonine protein kinases. PKCα is observed in the suprabasal layers of normal skin and is associated to keratinocyte growth arrest and differentiation processes. In hyper-proliferative skin diseases the presence of PKCα is altered. OBJECTIVE: The aim of the present study was to investigate valrubicin's mode of action in keratinocytes by studying its possible effect on PKCα activation. METHODS: PKCα's characteristic to translocate from the cytoplasm to the cellular membrane when activated was assessed by measuring the amount of PKCα in the soluble and membrane-bound protein fractions isolated from valrubicin stimulated keratinocytes and by visualizing PKCα in stimulated cells over time. Downstream signaling was investigated by measuring the amount of phosphorylated Myristoylated Alanine-rich C-kinase substrate (MARCKS) and extracellular signal-regulated kinases (ERK) 1/2 of valrubicin-stimulated keratinocytes. To investigate whether there was a direct interaction between valrubicin and PKCα, an activity assay employing purified PKCα protein was used. RESULTS: Valrubicin activates PKCα in vitro as shown by PKCα's translocation and phosphorylation of downstream signaling molecules. CONCLUSION: Valrubicin stimulates PKCα activity and downstream signaling which may contribute to its beneficial effect in psoriasis and NMSC.

A lentiviral vector-based genetic sensor system for comparative analysis of permeability and activity of vitamin D3 analogues in xenotransplanted human skin.

Vitamin D3 analogues are widely used topical and oral remedies for various ailments such as psoriasis, osteoporosis and secondary hyperparathyroidism. In topical treatment, high skin permeability and cellular uptake are key criteria for beneficial effects due to the natural barrier properties of skin. In this study, we wish to establish an in vivo model that allows the comparison of permeability and activity of vitamin D3 analogues in human skin. We generate a bipartite, genetic sensor technology that combines efficient lentivirus-directed gene delivery to xenotransplanted human skin with vitamin D3-induced expression of a luciferase reporter gene and live imaging of animals by bioluminescence imaging. Based on the induction of a transcriptional activator consisting of the vitamin D receptor fused to the Gal4 DNA-binding domain, the vitamin D3-responsive sensor facilitates non-invasive and rapid assessment of permeability and functional properties of vitamin D3 analogues. By topical application of a panel of vitamin D3 analogues onto 'sensorized' human skin, the sensor produces a drug-induced readout with a magnitude and persistence that allow a direct comparative analysis of different analogues. This novel genetic tool has great potential as a non-invasive in vivo screening system for further development and refinement of vitamin D3 analogues.

Resolution of psoriasis by a leukocyte-targeting bacterial protein in a humanized mouse model.

Psoriasis is a very common chronic skin disease, affecting 2-3% of the world's population or more than 125 million individuals worldwide. The characteristic lesion of psoriasis is due to rapid proliferation and shortened transition of keratinocytes through the epidermis. Proinflammatory white blood cells (WBCs) migrate into the psoriatic plaques, and the pathogenic cytokine environment causes the changes in keratinocyte proliferation and differentiation. Enhanced migration of WBCs is due to the upregulation and activation of adhesion molecules such as leukocyte function antigen-1 (LFA-1), which binds intercellular adhesion molecule-1 (ICAM-1) on endothelial cells. Targeting LFA-1 and preventing interaction with ICAM-1 has proven an effective strategy for treating psoriasis. We show here that a natural leukocyte-targeting bacterial protein (leukotoxin (LtxA)) that binds LFA-1 can inhibit proliferation of activated WBCs from psoriasis patients and demonstrates significant therapeutic efficacy in a psoriasis xenograft transplantation model. In ex vivo studies, LtxA preferentially targeted proinflammatory WBC subtypes, including activated CD25(+) T cells and CD14(+)CD16(+) monocytes. LFA-1 has been shown to have a significant role in the pathogenesis of numerous autoimmune and inflammatory diseases, and we propose that LtxA may be a highly effective agent for treating these diseases.

Targeting of human interleukin-12B by small hairpin RNAs in xenografted psoriatic skin.

BACKGROUND: Psoriasis is a chronic inflammatory skin disorder that shows as erythematous and scaly lesions. The pathogenesis of psoriasis is driven by a dysregulation of the immune system which leads to an altered cytokine production. Proinflammatory cytokines that are up-regulated in psoriasis include tumor necrosis factor alpha (TNFα), interleukin-12 (IL-12), and IL-23 for which monoclonal antibodies have already been approved for clinical use. We have previously documented the therapeutic applicability of targeting TNFα mRNA for RNA interference-mediated down-regulation by anti-TNFα small hairpin RNAs (shRNAs) delivered by lentiviral vectors to xenografted psoriatic skin. The present report aims at targeting mRNA encoding the shared p40 subunit (IL-12B) of IL-12 and IL-23 by cellular transduction with lentiviral vectors encoding anti-IL12B shRNAs. METHODS: Effective anti-IL12B shRNAs are identified among a panel of shRNAs by potency measurements in cultured cells. The efficiency and persistency of lentiviral gene delivery to xenografted human skin are investigated by bioluminescence analysis of skin treated with lentiviral vectors encoding the luciferase gene. shRNA-expressing lentiviral vectors are intradermally injected in xenografted psoriatic skin and the effects of the treatment evaluated by clinical psoriasis scoring, by measurements of epidermal thickness, and IL-12B mRNA levels. RESULTS: Potent and persistent transgene expression following a single intradermal injection of lentiviral vectors in xenografted human skin is reported. Stable IL-12B mRNA knockdown and reduced epidermal thickness are achieved three weeks after treatment of xenografted psoriatic skin with lentivirus-encoded anti-IL12B shRNAs. These findings mimic the results obtained with anti-TNFα shRNAs but, in contrast to anti-TNFα treatment, anti-IL12B shRNAs do not ameliorate the psoriatic phenotype as evaluated by semi-quantitative clinical scoring and by immunohistological examination. CONCLUSIONS: Our studies consolidate the properties of lentiviral vectors as a tool for potent gene delivery and for evaluation of mRNA targets for anti-inflammatory therapy. However, in contrast to local anti-TNFα treatment, the therapeutic potential of targeting IL-12B at the RNA level in psoriasis is questioned.

Statistical evaluation and experimental design of a psoriasis xenograft transplantation model treated with cyclosporin A.

Psoriasis xenograft transplantation models where human skin is transplanted onto immune-deficient mice are generally accepted in psoriasis research. Over the last decade, they have been widely employed to screen for new therapeutics with a potential anti-psoriatic effect. However, experimental designs differ in several parameters. Especially, the number of donors and grafts per experimental design varies greatly; numbers that are directly related to the probability of detecting statistically significant drug effects. In this study, we performed a statistical evaluation of the effect of cyclosporine A, a recognized anti-psoriatic drug, to generate a statistical model employable to simulate different scenarios of experimental designs and to calculate the associated statistical study power, defined as the probability of detecting a statistically significant anti-psoriatic drug treatment effect. Results showed that to achieve a study power of 0.8, at least 20 grafts per treatment group and a minimum of five donors should be included in the chosen experimental setting. To our knowledge, this is the first time that study power calculations have been performed to evaluate treatment effects in a psoriasis xenograft transplantation model. This study was based on a defined experimental protocol, thus other parameters such as drug potency, treatment protocol, mouse strain and graft size should, also, be taken into account when designing an experiment. We propose that the results obtained in this study may lend a more quantitative support to the validity of results obtained when exploring new potential anti-psoriatic drug effects.

Topical application of valrubicin has a beneficial effect on developing skin tumors.

Valrubicin is a second generation anthracycline characterized by an excellent safety profile presenting no skin toxicity or necrosis upon contact. In its current liquid formulation (Valstar; Indevus Pharmaceuticals, Lexington, MA), it is approved solely for the treatment of bladder cancer. Recently, valrubicin was incorporated in a cream formulation rendering this drug available for topical application. The cytostatic property of valrubicin can, thus, be employed for treating hyperproliferative skin diseases as was recently described for psoriasis. In the present study, the effect of topical application of valrubicin was investigated in skin tumor development; we hypothesized that valrubicin may be employed in treating actinic keratosis, a hyperproliferative skin condition that may transform into malignancy. A two-stage chemical mouse skin carcinogenesis model that represents the multistage etiology of human skin cancer-from developing papillomas to squamous cell carcinoma (SCC) was used. Moreover, two human skin SCC cell lines: DJM-1 and HSC-1 were cultured, to further investigate the effect of valrubicin in vitro. Cell viability was assessed by adenosine triphosphate presence, proliferation as proliferative cell nuclear antigen expression and apoptosis as cytokeratin 18 cleavage, caspase activation, poly-adenosine diphosphate-ribose-polymerase cleavage and bax and bcl-2 regulation. Valrubicin significantly inhibited tumor formation in the mouse skin carcinogenesis model and significantly decreased cell viability of the cultured human skin SCC cells. In both mouse skin and SCC cells, proliferation was significantly decreased. Apoptosis was significantly increased in SCC cells but unchanged in the treated mouse skin at study completion. This study demonstrated that topical application of valrubicin has a beneficial effect in treating developing skin tumors.

Valrubicin in a topical formulation treats psoriasis in a xenograft transplantation model.

Valrubicin is a cytostatic drug currently approved by the American Federal Drug Administration as a trademarked Valstar sterile solution for the treatment of bladder cancer. Valrubicin has shown an excellent therapeutic potential with minimal toxicity. This study investigated the effect in vivo of treating psoriasis with a daily topical application of valrubicin cream in a psoriasis xenograft transplantation model. Psoriasis is characterized by an accelerated keratinocyte proliferation, resulting in increased epidermal thickness. We thus studied the cytostatic potential of valrubicin on epidermal keratinocytes. In vivo, valrubicin treatment resulted in a normalization of epidermal morphology and a reduction in epidermal thickness after 12 days. In addition, the dermal vessel pattern was reduced and the stratum granulosum was regained. Staining for a regenerative proliferation marker showed a decrease in keratinocyte proliferation, and scattered epidermal cells showed apoptosis. In vitro, valrubicin was shown to localize solely to the cell cytoplasm in cultured keratinocytes and to reduce keratinocyte proliferation as well as increase apoptosis by activation of caspases 3, 7, and 9. Our results indicated that valrubicin successfully treats psoriasis in a xenograft transplantation model, suggesting that topical valrubicin may become an upcoming treatment for psoriasis.

Application of stereology to dermatological research.

Stereology is a set of mathematical and statistical tools to estimate three-dimensional (3-D) characteristics of objects from regular two-dimensional (2-D) sections. In medicine and biology, it can be used to estimate features such as cell volume, cell membrane surface area, total length of blood vessels per volume tissue and total number of cells. The unbiased quantification of these 3-D features allows for a better understanding of morphology in vivo compared with 2-D methods. This review provides an introduction to the field of stereology with specific emphasis on the application of stereology to dermatological research by supplying a short insight into the theoretical basis behind the technique and presenting previous dermatological studies in which stereology was an integral part. Both the theory supporting stereology and a practical approach in a dermatological setting are reviewed with the aim to provide the reader with the capability to better assess papers employing stereological estimators and to design stereological studies independently.

Amelioration of psoriasis by anti-TNF-alpha RNAi in the xenograft transplantation model.

Tumor necrosis factor-alpha (TNF-alpha) is upregulated in psoriatic skin and represents a prominent target in psoriasis treatment. The level of TNF-alpha-encoding mRNA, however, is not increased in psoriatic skin, and it remains unclear whether intervention strategies based on RNA interference (RNAi) are therapeutically relevant. To test this hypothesis the present study describes first the in vitro functional screening of a panel of short hairpin RNAs (shRNAs) targeting human TNF-alpha mRNA and, next, the transfer of the most potent TNF-alpha shRNA variant, as assessed in vitro, to human skin in the psoriasis xenograft transplantation model by the use of lentiviral vectors. TNF-alpha shRNA treatment leads to amelioration of the psoriasis phentotype in the model, as documented by reduced epidermal thickness, normalization of the skin morphology, and reduced levels of TNF-alpha mRNA as detected in skin biopsies 3 weeks after a single vector injection of lentiviral vectors encoding TNF-alpha shRNA. Our data show efficient lentiviral gene delivery to psoriatic skin and therapeutic applicability of anti-TNF-alpha shRNAs in human skin. These findings validate TNF-alpha mRNA as a target molecule for a potential persistent RNA-based treatment of psoriasis and establish the use of small RNA effectors as a novel platform for target validation in psoriasis and other skin disorders.

[Interleukin-20--a new target in psoriasis treatment]. / Interleukin 20--et nyt mål for psoriasisbehandling.

Interleukin-20 (IL-20) is suggested as a new target in psoriasis treatment. It was first described in 2001, and the potential role of this cytokine in psoriasis was suggested because mice which were over-expressing IL-20 developed a psoriasis-like phenotype of the skin. Subsequently, IL-20 expression levels were found to be increased in psoriasis skin, and it was observed that these levels normalized upon psoriasis treatment. In the psoriasis xenograft transplantation model, administration of IL-20 to non-lesional psoriasis skin transplanted onto immune-deficient mice demonstrated that IL-20 was involved in the psoriasis induction. More interestingly, improvement of psoriasis was induced by blocking IL-20 signaling.

Interleukin-20 as a target in psoriasis treatment.

Interleukin-20 (IL-20) is a new member of the IL-10 cytokine family discovered by a structural algorithm. IL-20 transgenic mice displayed skin abnormalities reminiscent of psoriasis, a finding that has prompted the investigation of this new interleukin in relation to this disease. This article reviews the role of IL-20 and its implication in psoriasis. It is shown that IL-20 and its receptors are found in human skin and that IL-20 is involved in proliferation, angiogenesis, and chemotaxis, all characteristics of psoriasis. We demonstrated that IL-20 induced the thickening of human epidermis in vivo; however, this thickening does not seem to be related to a direct effect of IL-20 on hyperproliferation since the growth of normal human epidermal keratinocytes (NHEKs) cultured in vitro was not affected by IL-20. On the other hand, in vitro, IL-20 stimulated human peripheral blood mononuclear cells (PBMCs) to produce proinflammatory cytokines and, in vivo, IL-20 in combination with PBMCs induced psoriasis. This may suggest that IL-20 indirectly exerts its proliferative effects on keratinocytes via immune cells present in the skin. Finally, we found that blocking IL-20 signaling in psoriasis improves psoriasis, suggesting that IL-20 is a potential target in psoriasis treatment.

Subcutaneous adipocytes can differentiate into bone-forming cells in vitro and in vivo.

Interconversion of bone marrow osteoblasts and adipocytes has been reported previously. However, the osteogenic potential of extramedullary adipocytes is not known. Thus, we incubated a pure culture of human subcutaneous adipocytes in control medium for 1-2 weeks. Afterward, the cells were incubated in either osteoblast medium (OB medium) containing various combinations of calcitriol, dexamethasone, ascorbic acid, and beta-glycerophosphate or in adipocyte medium (AD medium) containing HEPES, biotin, pantothenate, insulin, triiodothyronine, dexamethasone, and isobutylmethylxanthine for 4 weeks. Expression of osteoblastic and adipocytic phenotypes was examined by determination of lineage-specific mRNA markers and in vitro adipocyte and osteoblast formation. Cells were also implanted, mixed with hydroxyapatite-tricalcium phosphate powder, in the subcutaneous tissue of immunodeficient mice in order to assess in vivo bone formation potential. One week after incubation in control medium, cells formed fusiform elongated fibroblast-like cells. In OB medium, cells stained positive for alkaline phosphatase (AP) and expressed mRNAs encoding Cbfa1/Runx2, AP, and osteocalcin. In AD medium cells reacquired adipocyte morphology with multilocular lipid-filled cells. Also, the cells expressed adipocyte-specific mRNA markers: lipoprotein lipase and peroxisome proliferator-activated receptor gamma2. Bone was formed only in the in vivo implants of cells incubated in OB medium. In conclusion, extramedullary adipocytes can transdifferentiate to bone-forming cells. Because of their ease of isolation, adipocytes may be good candidates for tissue-engineering protocols aimed at creating bone tissue for the repair of nonunion fractures and large bone defects.

Mice deficient in 11beta-hydroxysteroid dehydrogenase type 1 lack bone marrow adipocytes, but maintain normal bone formation.

Glucocorticoids (GCs) exert potent, but poorly characterized, effects on the skeleton. The cellular activity of GCs is regulated at a prereceptor level by 11beta-hydroxysteroid dehydrogenases (11betaHSDs). The type 1 isoform, which predominates in bone, functions as a reductase in intact cells and regenerates active cortisol (corticosterone) from circulating inert 11-keto forms. The aim of the present study was to investigate the role of this intracrine activation of GCs on normal bone physiology in vivo using mice deficient in 11betaHSD1 (HSD1(-/-)). The HSD1(-/-) mice exhibited no significant changes in cortical or trabecular bone mass compared with wild-type (Wt) mice. Aged HSD1(-/-) mice showed age-related bone loss similar to that observed in Wt mice. Histomorphometric analysis showed similar bone formation and bone resorption parameters in HSD1(-/-) and Wt mice. However, examination of bone marrow composition revealed a total absence of marrow adipocytes in HSD1(-/-) mice. Cells from Wt and HSD1(-/-) mice exhibited similar growth rates as well as similar levels of production of osteoblastic markers. The adipocyte-forming capacity of in vitro cultured bone marrow stromal cells and trabecular osteoblasts was similar in HSD1(-/-) and Wt mice. In conclusion, our results suggest that 11betaHSD1 amplification of intracellular GC actions in mice may be required for bone marrow adipocyte formation, but not for bone formation. The clinical relevance of this observation remains to be determined.