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Precise classification of sarcomas is crucial to optimal clinical management. In this prospective, multicenter, observational study within the Hellenic Group of Sarcoma and Rare Cancers (HGSRC), we assessed the effect of expert pathology review, coupled with the application of molecular diagnostics, on the diagnosis and management of sarcoma patients. Newly diagnosed sarcoma patients were addressed by their physicians to one of the two sarcoma pathologists of HGSRC for histopathological diagnostic assessment. RNA next-generation sequencing was performed on all samples using a platform targeting 86 sarcoma gene fusions. Additional molecular methods were performed in the opinion of the expert pathologist. Therefore, the expert pathologist provided a final diagnosis based on the histopathological findings and, when necessary, molecular tests. In total, 128 specimens from 122 patients were assessed. Among the 119 cases in which there was a preliminary diagnosis by a non-sarcoma pathologist, there were 37 modifications in diagnosis (31.1%) by the sarcoma pathologist, resulting in 17 (14.2%) modifications in management. Among the 110 cases in which molecular tests were performed, there were 29 modifications in diagnosis (26.4%) through the genomic results, resulting in 12 (10.9%) modifications in management. Our study confirms that expert pathology review is of utmost importance for optimal sarcoma diagnosis and management and should be assisted by molecular methods in selected cases.
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BACKGROUND/AIM: Uterine leiomyosarcomas (uLMS) are the most common mesenchymal tumors of the female genital tract. uLMS genetics encompass complex karyotypes with no specific molecular alterations. The Hippo pathway has been implicated in the pathogenesis of epithelioid hemangio-endotheliomas and endometrial sarcomas. Hippo pathway effectors are YAP1 and TAZ co-transcriptional factors. PATIENTS AND METHODS: We studied Hippo pathway in a series of 32 uLMS patients and its association with clinicopathological parameters. MATERIALS AND METHODS: Immunohistochemical analysis of YAP1 and TAZ proteins accompanied with fluorescent in situ hybridization study of YAP1 gene was performed in patient samples. Age, sex, tumor size, stage at the time of diagnosis and treatment have been analyzed. Overall survival (OS) was calculated from the time of diagnosis until death, loss of follow up or data cut-off. RESULTS: Hippo signaling was found to be dysregulated in 20 (62.5%) patients with uLMS. Regarding OS we detected a trend of Hippo deregulation, designating it as a positive prognostic factor. CONCLUSION: The Hippo pathway is implicated in uLMS oncogenesis, since nuclear expression of YAP1 was detected in 17 (53.1%) of the 32 patients with immunohistochemistry and YAP1 amplification was found in 8 (25%) patients.
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Proteínas Adaptadoras Transductoras de Señales , Vía de Señalización Hippo , Leiomiosarcoma , Proteínas Serina-Treonina Quinasas , Transducción de Señal , Factores de Transcripción , Neoplasias Uterinas , Proteínas Señalizadoras YAP , Humanos , Femenino , Leiomiosarcoma/genética , Leiomiosarcoma/patología , Leiomiosarcoma/metabolismo , Neoplasias Uterinas/patología , Neoplasias Uterinas/metabolismo , Neoplasias Uterinas/genética , Persona de Mediana Edad , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Señalizadoras YAP/metabolismo , Proteínas Señalizadoras YAP/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Anciano , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Adulto , Pronóstico , Inmunohistoquímica , Hibridación Fluorescente in Situ , Regulación Neoplásica de la Expresión Génica , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZRESUMEN
Introduction A constant infusion of local anesthetics through pain pumps has been shown to cause chondrolysis. However, there is no general consensus regarding the safety of a single intra-articular injection of local anesthetics. In this experimental study, we examined the rat cartilage for possible histological effects after a single intra-articular administration of lidocaine or ropivacaine. Material and methods Thirty-two male Sprague-Dawley rats, weighing 250-300 grams, were divided into two groups of 16 each. We injected 0.1 ml of either lidocaine 2% (20 mg/ml) or ropivacaine 0.75% (7.5 mg/ml) into the left knee of the rats. The right knee in both groups was used as a control, and an equal amount of normal saline was injected. Each group was further divided into subgroups of four, which were euthanized after one, seven, 21, and 60 days after the initial injection. Knees were excised and prepared for histopathological analysis. A modified version of the Mankin score was used for cartilage damage evaluation. Results No difference regarding cartilage damage was detected after the examination under light microscopy between lidocaine, ropivacaine, and placebo in all specimens. Time elapsed since the initial injection did not affect the results at any time point. Conclusion A single intra-articular injection of local anesthetic did not induce any histological changes in the rat cartilage. Further research is needed to demonstrate the safety of humans.
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The calcium-selective ion channel Orai1 has a complex role in bone homeostasis, with defects in both bone production and resorption detected in Orai1 germline knock-out mice. To determine whether Orai1 has a direct, cell-intrinsic role in osteoblast differentiation and function, we bred Orai1 flox/flox (Orai1fl/fl) mice with Runx2-cre mice to eliminate its expression in osteoprogenitor cells. Interestingly, Orai1 was expressed in a mosaic pattern in Orai1fl/fl-Runx2-cre bone. Specifically, antibody labeling for Orai1 in vertebral sections was uniform in wild type animals, but patchy regions in Orai1fl/fl-Runx2-cre bone revealed Orai1 loss while in other areas expression persisted. Nevertheless, by micro-CT, bones from Orai1fl/fl-Runx2-cre mice showed reduced bone mass overall, with impaired bone formation identified by dynamic histomorphometry. Cortical surfaces of Orai1fl/fl-Runx2-cre vertebrae however exhibited patchy defects. In cell culture, Orai1-negative osteoblasts showed profound reductions in store-operated Ca2+ entry, exhibited greatly decreased alkaline phosphatase activity, and had markedly impaired substrate mineralization. We conclude that defective bone formation observed in the absence of Orai1 reflects an intrinsic role for Orai1 in differentiating osteoblasts.
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Canales de Calcio , Subunidad alfa 1 del Factor de Unión al Sitio Principal , Osteoblastos , Animales , Ratones , Calcio/metabolismo , Canales de Calcio/genética , Canales de Calcio/metabolismo , Subunidad alfa 1 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Ratones Noqueados , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Osteoblastos/metabolismoRESUMEN
BACKGROUND: Long bone fractures display significant non-union rates, but the exact biological mechanisms implicated in this devastating complication remain unclear. The combination of osteogenetic and angiogenetic factors at the fracture site is an essential prerequisite for successful bone regeneration. The aim of this study is to investigate the results of the clinical implantation of growth factors for intraoperative enhancement of osteogenesis for the treatment of long bone fractures and non-unions. METHODS: A systematic literature review search was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines in the PubMed and Web of Science databases from the date of inception of each database through to 10 January 2022. Specific inclusion and exclusion criteria were applied in order to identify relevant studies reporting on the treatment of upper and lower limb long bone non-unions treated with osteoinductive or cellular factors. RESULTS: Overall, 18 studies met the inclusion criteria and examined the effectiveness of the application of Bone Morphogenetic Proteins-2 and -7 (BMPs), platelet rich plasma (PRP) and mesenchymal stem cells (MSCs). Despite the existence of limitations in the studies analysed (containing mixed groups of open and close fractures, different types of fractures, variability of treatment protocols, different selection criteria and follow-up periods amongst others), their overall effectiveness was found significantly increased in patients who received them compared with the controls (I2 = 60%, 95% CI = 1.59 [0.99-2.54], Z =1.93, p = 0.05). CONCLUSION: Administration of BMP-2 and -7, PRP and MSCs were considered effective and safe methods in fracture treatment, increasing bone consolidation, reducing time to repair and being linked to satisfactory postoperative functional scores.
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Dickkopf-1 (Dkk-1) is a key regulator of bone remodeling in spondyloarthropathies. Nevertheless, data regarding its expression in cells of pathophysiologic relevance, such as mesenchymal stem cells (MSCs), are lacking. Herein, we aimed to address DKK1 gene expression and Wnt pathway activation in MSCs from patients with ankylosing spondylitis (AS) and explore the effect of IL-17 on MSCs with respect to DKK-1 expression and Wnt pathway activation. Primary MSCs were isolated from the bone marrow of the femoral head of two patients with AS and two healthy controls undergoing orthopedic surgery. MSCs were cultured for 7 days in expansion medium and for 21 days in osteogenic medium in the presence or absence of IL-17A. Gene expression of DKK-1 and osteoblastic markers was determined by RT-PCR. Alkaline phosphatase activity, alizarin red and Van Kossa staining were used to assess osteoblastic function and mineralization capacity. DKK-1 was significantly downregulated in MSCs and osteoblasts from patients with AS compared to controls. Moreover, MSCs and osteoblasts from AS patients displayed increased Wnt pathway activation and enhanced osteoblastic activity, as indicated by increased expression of osteoblast marker genes and alkaline phosphatase activity. IL-17 downregulated DKK-1 expression and increased osteoblastic activity and mineralization capacity. DKK-1 is underexpressed in MSCs from AS patients compared to controls, whereas IL-17 has an inhibitory effect on DKK-1 expression and stimulates osteoblastic function. These data may have pathogenetic and clinical implications in AS.
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Péptidos y Proteínas de Señalización Intercelular/metabolismo , Células Madre Mesenquimatosas , Espondilitis Anquilosante , Fosfatasa Alcalina/metabolismo , Diferenciación Celular , Humanos , Interleucina-17/metabolismo , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/metabolismo , Osteogénesis , Proteínas/metabolismo , Espondilitis Anquilosante/metabolismo , Vía de Señalización WntRESUMEN
In the present study, we studied the effect of apolipoprotein A-1 (APOA1) on the spatial and molecular characteristics of bone marrow adipocytes, using well-characterized ApoA1 knockout mice. APOA1 is a central regulator of high-density lipoprotein cholesterol (HDL-C) metabolism, and thus HDL; our recent work showed that deficiency of APOA1 increases bone marrow adiposity in mice. We found that ApoA1 deficient mice have greatly elevated adipocytes within their bone marrow compared to wild type counterparts. Morphologically, the increased adipocytes were similar to white adipocytes, and displayed proximal tibial-end localization. Marrow adipocytes from wild type mice were significantly fewer and did not display a bone-end distribution pattern. The mRNA levels of the brown/beige adipocyte-specific markers Ucp1, Dio2, Pat2, and Pgc1a; and the expression of leptin were greatly reduced in the ApoA1 knock-out in comparison to the wild-type mice. In the knock-out mice, adiponectin was remarkably elevated. In keeping with the close ties of hematopoietic stem cells and marrow adipocytes, using flow cytometry we found that the elevated adiposity in the ApoA1 knockout mice is associated with a significant reduction in the compartments of hematopoietic stem cells and common myeloid, but not of the common lymphoid, progenitors. Moreover, the 'beiging'-related marker osteopontin and the angiogenic factor VEGF were also reduced in the ApoA1 knock-out mice, further supporting the notion that APOA1-and most probably HDL-C-regulate bone marrow microenvironment, favoring beige/brown adipocyte characteristics.
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Adipocitos Beige , Apolipoproteína A-I , Adipocitos Beige/metabolismo , Adipocitos Blancos/metabolismo , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/metabolismo , Animales , Apolipoproteína A-I/genética , Apolipoproteína A-I/metabolismo , Médula Ósea/metabolismo , Ratones , Ratones Noqueados , Obesidad/metabolismoRESUMEN
The purpose of the present study was to analyze normal and degenerated menisci with Raman methodology on thin sections of formalin fixed paraffin embedding tissues and to correlate the Raman findings with the grade of meniscus degeneration. Menisci (n = 27) were removed from human knee joints after total knee replacement or meniscectomy. Following routine histopathological analysis to determine the grade of meniscal lesions obtained from healthy and degenerated formaline fixed paraffin embedded (FFPE) meniscal sections, Raman polarization approach was applied to evaluate the orientation of collagen fibrils in different levels of the same 5 µm thick FFPE meniscal tissue sections, used for histopathological assessment. We collected Raman spectra in two different polarization geometries, v-HH and v-VV, and calculated the mean value of the v-HH/v-VV intensity ratio of two Raman bands, sensitive and non-sensitive to the molecular orientation. The collagen specific amide I band at 1665 cm-1, has the higher sensitivity dependence on the Raman polarization. The mean values of ratio v-HH/v-VV of the 1665 cm-1 peak intensity was significantly higher in healthy, mean ± SD: 2.56 ± 0.46, compared to degenerated menisci, mean ± SD: 1.85 ± 0.42 (p = 0.0014). The mean values of v-HH/v-VV intensity ratio were 2.18 and 1.50 for low and high degenerated menisci, respectively (p < 0.0001). The difference of peak intensities in the two laser polarizations is decreased in the degenerated meniscus; this difference is diminishing as the degeneration increases. The v-HH/v-VV ratio was also of significant difference in low as compared to control and high grade meniscus lesions (p = 0.036 and p < 0.0001, respectively) offering valuable information for the approach of its biology and function. In the present study we showed that the 5 µm thick sections can be used for Raman analysis of meniscal tissue with great reliability, in terms of sensitivity, specificity, false-negative and false-positive results. Our data introduce the interesting hypothesis that compact portable Raman microscopy on tissue sections can be used intra-operatively for fast diagnosis and hence, accurate procedure design in the operating room.
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Colágeno/química , Meniscos Tibiales/fisiopatología , Espectrometría Raman/métodos , Adulto , Anciano , Anciano de 80 o más Años , Algoritmos , Artroplastia de Reemplazo de Rodilla , Diagnóstico Diferencial , Matriz Extracelular , Reacciones Falso Positivas , Femenino , Humanos , Masculino , Meniscectomía , Menisco/cirugía , Microscopía , Persona de Mediana Edad , Ortopedia , Osteoartritis de la Rodilla/patología , Parafina/química , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Fracture-healing is a complex multi-stage process that usually progresses flawlessly, resulting in restoration of bone architecture and function. Regrettably, however, a considerable number of fractures fail to heal, resulting in delayed unions or non-unions. This may significantly impact several aspects of a patient's life. Not surprisingly, in the past few years, a substantial amount of research and number of clinical studies have been designed, aiming at shedding light into the cellular and molecular mechanisms that regulate fracture-healing. Herein, we present the current knowledge on the pathobiology of the fracture-healing process. In addition, the role of skeletal cells and the impact of marrow adipose tissue on bone repair is discussed. Unveiling the pathogenetic mechanisms that govern the fracture-healing process may lead to the development of novel, smarter, and more effective therapeutic strategies for the treatment of fractures, especially of those with large bone defects.
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An adult man presented with a 5-month history of anterior right shoulder pain. He denied previous trauma or night pain. On the otherwise normal physical examination, pain was elicited in maximum abduction and external rotation. Radiographs were negative. The primary imaging findings were bone marrow oedema of the inferomedial proximal metaphysis of the humerus on MRI and cortical demineralisation on CT located posteromedially. A superficial elevation was also observed around the lesion. A provisional diagnosis of an osteoid osteoma was made based on these imaging findings. Arthroscopic excision of the lesion was performed, and histopathological examination confirmed the diagnosis of an osteoid osteoma. Diagnosis of intra-articular osteoid osteomas may be challenging due to atypical symptomatology and lack of pathognomonic imaging findings. Arthroscopic excision of such lesions in the shoulder is a safe and reliable option and should be considered as the treatment of choice.
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Neoplasias Óseas , Osteoma Osteoide , Adulto , Artroscopía , Neoplasias Óseas/diagnóstico , Humanos , Húmero/diagnóstico por imagen , Masculino , Osteoma Osteoide/diagnóstico , Hombro/diagnóstico por imagen , Dolor de Hombro/etiologíaRESUMEN
Advances in 3D bioprinting have allowed the use of stem cells along with biomaterials and growth factors toward novel tissue engineering approaches. However, the cost of these systems along with their consumables is currently extremely high, limiting their applicability. To address this, we converted a 3D printer into an open source 3D bioprinter and produced a customized bioink based on accessible alginate/gelatin precursors, leading to a cost-effective solution. The bioprinter's resolution, including line width, spreading ratio and extrusion uniformity measurements, along with the rheological properties of the bioinks were analyzed, revealing high bioprinting accuracy within the printability window. Following the bioprinting process, cell survival and proliferation were validated on HeLa Kyoto and HEK293T cell lines. In addition, we isolated and 3D bioprinted postnatal neural stem cell progenitors derived from the mouse subventricular zone as well as mesenchymal stem cells derived from mouse bone marrow. Our results suggest that our low-cost 3D bioprinter can support cell proliferation and differentiation of two different types of primary stem cell populations, indicating that it can be used as a reliable tool for developing efficient research models for stem cell research and tissue engineering.
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We investigated the effect of a phytoestrogen, (3R)-1,7-diphenyl-(4E,6E)-4,6-heptadien-3-ol (DPHD), from Curcuma comosa Roxb. (Zingiberaceae family) on the adipogenic differentiation of mesenchymal progenitors, human bone marrow-derived mesenchymal stem cells (hBMSCs). DPHD inhibited adipocyte differentiation of hBMSCs by suppressing the expression of genes involved in adipogenesis. DPHD at concentrations of 0.1, 1, and 10 µM significantly decreased triglyceride accumulation in hBMSCs to 7.1 ± 0.2, 6.3 ± 0.4, and 4.9 ± 0.2 mg/dL, respectively, compared to the nontreated control (10.1 ± 0.9 mg/dL) (p < 0.01). Based on gene expression profiling, DPHD increased the expression of several genes involved in the Wnt/ß-catenin signaling pathway, a negative regulator of adipocyte differentiation in hBMSCs. DPHD also increased the levels of essential signaling proteins which are extracellular signal-regulated kinases 1 and 2 (ERK1/2) and glycogen synthase kinase 3 beta (GSK-3ß) that link estrogen receptor (ER) signaling to Wnt/ß-catenin signaling. In conclusion, DPHD exhibited the anti-adipogenic effect in hBMSCs by suppression of adipogenic markers in hBMSCs through the activation of ER and Wnt/ß catenin signaling pathways. This finding suggests the potential role of DPHD in preventing bone marrow adiposity which is one of the major factors that exacerbates osteoporosis in postmenopause.
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Adipocitos/efectos de los fármacos , Adipogénesis/efectos de los fármacos , Curcuma/química , Diarilheptanoides/farmacología , Células Madre Mesenquimatosas/efectos de los fármacos , Fitoestrógenos/farmacología , Extractos Vegetales/farmacología , Adipocitos/citología , Adipocitos/metabolismo , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Células de la Médula Ósea/metabolismo , Diferenciación Celular/efectos de los fármacos , Diarilheptanoides/química , Glucógeno Sintasa Quinasa 3 beta/genética , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Osteoblastos/metabolismo , Fitoestrógenos/química , Extractos Vegetales/química , Receptores de Estrógenos/genética , Receptores de Estrógenos/metabolismo , Triglicéridos/metabolismo , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismoRESUMEN
Introduction Platelet-rich plasma (PRP) products and mesenchymal stem cells (MSCs) seem to have a significant potential as neurogenic therapeutic modulator systems. This study aimed to investigate such biological blood derivatives that could enhance nerve regeneration when applied locally in the primary repair of peripheral nerve transection of an experimental rat model. Methods A total of 42 two-month-old male Wistar rats were divided into three "treatment" groups (control, PRP, and MSCs). All the subjects were operated under anesthesia, and the surgical site was infiltrated with either normal saline, PRP derived from the animal's peripheral blood, or MSCs derived from the animal's femoral bone marrow. All three groups were also sub-divided into two sub-groups based on the post-operative administration of Non-steroidal anti-inflammatory drugs (NSAIDs) or not in order to evaluate the effect of NSAIDs on the final outcome. Three months post-surgery, electromyography evaluation of both hind limbs (right operated and left non-operated) was performed. The animals were euthanized, and nerve repair specimens were prepared for histology. Results PRP group had a significant effect (p<0.05) on the sciatic nerve repair when compared with the control group, whereas the MSC group had a positive effect but was not statistically significant (p=0.2). The number of counted neural axons at the area distal to the nerve repair site were significantly repetitive (p<0.05) in both the PRP and MSC groups when compared with the control group. Conclusions Both PRP and MSCs appear to play an essential role in the enhancement of nerve repair in terms of functionality and histology. MSCs group demonstrated a positive effect, whereas the PRP group showed statistically significant better results.
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OBJECTIVE: To assess: (i) the prevalence, and clinical and imaging characteristics of immune checkpoint inhibitor (ICI)-induced musculoskeletal immune-related adverse events (ir-AEs) in a prospective manner and (ii) whether serum levels of cytokines associated with the Th1/Th2/Th17 response are differentially expressed in patients with and without musculoskeletal Ir-AEs. METHODS: All patients treated with ICI who developed musculoskeletal manifestations were referred to the Rheumatology Department, and an MRI of the involved area(s) was performed. RESULTS: During the study period, a total of 130 patients were treated with ICIs. Of these, 10 (7.7%) developed ICI-induced Ir-AEs. The median time from ICI treatment since development of symptoms was 2.5 months. Three different patterns of musculoskeletal manifestations were found: (i) prominent joint involvement (n = 3); (ii) prominent 'periarticular' involvement (n = 4). These patients had diffuse swelling of the hands, feet or knees. MRI depicted mild synovitis with more prominent myositis and/or fasciitis in the surrounding tissues in all cases; (iii) myofasciitis (n = 3). Clinically, these patients presented with pain in the knee(s)/thigh(s), whereas MRI depicted myofasciitis of the surrounding muscles. Patients with musculoskeletal ir-AEs had significantly higher oncologic response rates compared with patients not exhibiting musculoskeletal ir-AEs (50% vs 12.5%, respectively, P = 0.0016). Cytokine levels associated with a Th1/Th2/Th17 response were similar between patients with and without musculoskeletal ir-AEs. Overall, symptoms were mild/moderate and responded well to treatment, with no need for ICI discontinuation. CONCLUSION: In our cohort, ICI-induced musculoskeletal manifestations developed in 7.7% of patients. Imaging evidence of myofasciitis was found in most patients, indicating that the muscle/fascia is more frequently involved than the synovium.
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Antineoplásicos Inmunológicos/efectos adversos , Factores Inmunológicos/efectos adversos , Imagen por Resonancia Magnética/métodos , Enfermedades Musculoesqueléticas/inducido químicamente , Enfermedades Reumáticas/inducido químicamente , Antineoplásicos Inmunológicos/administración & dosificación , Estudios de Cohortes , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Esquema de Medicación , Fascitis/inducido químicamente , Fascitis/diagnóstico por imagen , Fascitis/epidemiología , Femenino , Humanos , Factores Inmunológicos/administración & dosificación , Incidencia , Masculino , Persona de Mediana Edad , Enfermedades Musculoesqueléticas/diagnóstico por imagen , Enfermedades Musculoesqueléticas/epidemiología , Miositis/inducido químicamente , Miositis/diagnóstico por imagen , Miositis/epidemiología , Estudios Prospectivos , Enfermedades Reumáticas/diagnóstico por imagen , Enfermedades Reumáticas/epidemiología , Índice de Severidad de la EnfermedadRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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We examined bone formation and turnover in high-density lipoprotein (HDL) receptor, scavenger receptor type I (Scarb1), knockout animals relative to wild-type (WT) controls. Scarb1-/- animals have elevated serum adrenocorticotropic hormone (ACTH) due to the role of Scarb1 in glucocorticoid production, which might cause increased bone mass. However, this was not observed: Scarb1-/- mice, with ACTH, over 1000 pg/ml relative to wild-type ACTH ~ 25 pg/ml, bone of the knockout animals was osteopenic relative to the wild type at 16 weeks, including bone volume/total volume and trabecular thickness. Other serum parameters of WT and Scarb1-/- animals in cortisol or calcium were unaffected, although Scarb1-/- animals had significantly elevated PTH and decreased phosphate. Osteoblast and osteoclast-related mRNAs extracted from bone were greatly decreased at 8 or 16 weeks. Importantly, in normal ACTH, osteogenic differentiation in vitro from mesenchymal stem cells showed reduced alkaline phosphatase and mineralization. In Scarb1-/- cells relative to WT, mRNAs for RunX2, alkaline phosphatase, type I collagen, and osteocalcin were reduced 40-90%, all p < 0.01, indicating a role of Scarb1 in osteoblast differentiation independent of ACTH. Additionally, in vitro osteoblast differentiation at variable ACTH in WT cells confirmed ACTH increasing bone differentiation, mineralization, alkaline phosphatase, and osteocalcin mRNA at 0-10 nM ACTH, but reduced bone differentiation at 100-1000 nM ACTH. Overall Scarb1-/- animals show inhibited bone formation with age. This may be a mixed effect on direct bone formation and of very high ACTH. Further, this work shows that both ACTH concentration and the HDL receptor Scarb1 play important independent roles in osteoblast differentiation.
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Hormona Adrenocorticotrópica/sangre , Diferenciación Celular , Osteoblastos , Osteogénesis , Receptores Depuradores de Clase B/fisiología , Animales , Densidad Ósea , Remodelación Ósea , Femenino , Masculino , Ratones , Ratones Noqueados , Osteoclastos , Cultivo Primario de CélulasRESUMEN
BACKGROUND: Uncoupling protein 1 (UCP1) is a mitochondral protein transporter that uncouples electron transport from ATP production. UCP1 is highly expressed in brown adipose tissue (BAT), including hibernomas, but its expression in other adipose tumours is uncertain. UCP1 has also been found in other tissues (e.g. smooth muscle) but whether it is expressed in non-adipose benign and malignant soft tissue tumours is unknown. METHODS: Immunohistochemical staining of normal (axillary) BAT and subcutaneous/abdominal white adipose tissue (WAT) as well as a wide range of benign and malignant primary soft tissue tumours (n = 171) was performed using a rabbit polyclonal antibody to UCP1. BAT and hibernomas were also stained by immunohistochemistry with monoclonal and polyclonal antibodies to adipose/non-adipose tumour markers in order to characterise the immunophenotype of BAT cells. RESULTS: UCP1 was strongly expressed in the cytoplasm of brown fat cells in BAT and hibernomas, both of which also expressed aP2, S100, CD31, vimentin and calponin. UCP1 was not expressed in WAT or other adipose tumours with the exception a few tumour cells in pleomorphic liposarcoma. UCP1 was variably expressed by tumour cells in a few non-adipose sarcomas including leiomyosarcoma, rhabdomyosarcoma, alveolar soft part sarcoma, synovial sarcoma and clear cell sarcoma. CONCLUSIONS: UCP1 is strongly expressed in BAT but not WAT and is found in all hibernomas and a few pleomorphic liposarcomas but not in other adipose tumours. UCP1 expression in a few non-adipose soft tissue sarcomas may possibly reflect origin of tumour cells from a common mesenchymal stem cell precursor and/or developmental pathway.
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Studying human cancer from a biomechanical perspective may contribute to pathogenesis understanding which leads to the malignancy. In this study, biomechanics of suspended and adhered breast cancer cells were investigated via the micropipette aspiration method with special emphasis on comparing the cell stiffness and viscoelastic parameters of estrogen receptor positive, ER+, MCF-7 and human epidermal growth factor receptor 2 positive, HER2 +, SKBR-3 cancer cell lines prior to and post treatment with tamoxifen and trastuzumab, respectively. Alterations of mechanical parameters included significant increase in cell stiffness, especially after treatment with trastuzumab and changes in viscoelastic parameters, in both cancer cell lines post treatment. According to immunofluorescence analysis, the raised cell stiffness was corresponded to remodeling of F-actin, which peripherally located in tamoxifen treated and perinuclear accumulated in trastuzumab treated cancer cell cytoskeleton, implying a reduced potential for cell deformation and motility. Additionally, these results were in line with the study of single and collective cell migration through Boyden chamber and wound healing assays respectively, where the potential for migration was significantly decreased after treatment. Consequently, these findings lead to an increased interest in biomechanics of cancer progression after treatment with anti-tumor agents, importantly in understanding the effect of the alterations of mechanical properties upon the possibility for change in metastatic potential.
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Antineoplásicos/farmacología , Neoplasias de la Mama/patología , Tamoxifeno/farmacología , Trastuzumab/farmacología , Citoesqueleto de Actina/química , Actinas/química , Línea Celular Tumoral , Elasticidad , Humanos , Células MCF-7 , ViscosidadRESUMEN
Biglycan, a small leucine rich proteoglycan (SLRP), is an important participant in bone homeostasis and development as well as in bone pathology. In the present study biglycan was identified as a positive regulator of MG63 osteosarcoma cell growth (p ≤ 0.001). IGF-I was shown to increase biglycan expression (p ≤ 0.01), whereas biglycan-deficiency attenuated significantly both basal and IGF-I induced cell proliferation of MG63 cells (p ≤ 0.001; p ≤ 0.01, respectively). These effects were executed through the IGF-IR receptor whose activation was strongly attenuated (p ≤ 0.01) in biglycan-deficient MG63 cells. Biglycan, previously shown to regulate Wnt/ß-catenin pathway, was demonstrated to induce a significant increase in ß-catenin protein expression evident at cytoplasmic (p ≤ 0.01), membrane (p ≤ 0.01), and nucleus fractions in MG63 cells (p ≤ 0.05). As demonstrated by immunofluorescence, increase in ß-catenin expression is attributed to co-localization of biglycan with the Wnt co-receptor low-density lipoprotein receptor-related protein 6 (LRP6) resulting in attenuated ß-catenin degradation. Furthermore, applying anti-ß-catenin and anti-pIGF-IR antibodies to MG-63 cells demonstrated a cytoplasmic and to the membrane interaction between these molecules that increased upon exogenous biglycan treatment. In parallel, the downregulation of biglycan significantly inhibited both basal and IGF-I-dependent ERK1/2 activation, (p ≤ 0.001). In summary, we report a novel mechanism where biglycan through a LRP6/ß-catenin/IGF-IR signaling axis enhances osteosarcoma cell growth.
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OBJECTIVES: The activity of the Wnt pathway, a critical mediator of fibrosis, is regulated by Dickkopf-1 (Dkk-1). Dkk-1 is absent from scleroderma skin in contrast to skin from healthy subjects where it is clearly expressed. There are no data on circulating levels and function of Dkk-1 in patients with systemic sclerosis (SSc). Our objectives are to assess: i) circulating and functional levels of Dkk-1 in patients with SSc and ii) whether the striking lack of Dkk-1 skin expression is also evident in a) clinically uninvolved skin from patients with SSc and b) very early disease prior to skin thickening. METHODS: Circulating Dkk-1 levels were measured in 50 patients with SSc and 50 controls. Skin biopsies were obtained from SSc patients from a) clinically involved skin b) clinically uninvolved skin, c) oedematous skin prior to skin thickening. RESULTS: Circulating and functional Dkk-1 levels were similar in patients with SSc and controls. Healthy skin displayed a high Dkk-1 immuno-expression in the epidermis and dermal fibroblasts in contrast to clinically involved scleroderma skin where Dkk-1 was totally absent. In all biopsies of clinically uninvolved skin Dkk-1 was only moderately expressed whereas skin from very early disease displayed only a weak Dkk-1 immunoreactivity. CONCLUSIONS: The downregulation of Dkk-1 at the oedematous phase of the disease indicates that the Wnt pathway is involved early in the disease process and may play a role in driving fibrosis. The decrease in Dkk-1 expression in clinically uninvolved scleroderma skin indicates that skin in SSc is universally affected.