Silencing NTPDase3 activity rehabilitates the osteogenic commitment of post-menopausal stem cell bone progenitors.

BACKGROUND: Endogenously released adenine and uracil nucleotides favour the osteogenic commitment of bone marrow-derived mesenchymal stromal cells (BM-MSCs) through the activation of ATP-sensitive P2X7 and UDP-sensitive P2Y6 receptors. Yet, these nucleotides have their osteogenic potential compromised in post-menopausal (Pm) women due to overexpression of nucleotide metabolizing enzymes, namely NTPDase3. This prompted us to investigate whether NTPDase3 gene silencing or inhibition of its enzymatic activity could rehabilitate the osteogenic potential of Pm BM-MSCs. METHODS: MSCs were harvested from the bone marrow of Pm women (69 ± 2 years old) and younger female controls (22 ± 4 years old). The cells were allowed to grow for 35 days in an osteogenic-inducing medium in either the absence or the presence of NTPDase3 inhibitors (PSB 06126 and hN3-B3s antibody); pre-treatment with a lentiviral short hairpin RNA (Lenti-shRNA) was used to silence the NTPDase3 gene expression. Immunofluorescence confocal microscopy was used to monitor protein cell densities. The osteogenic commitment of BM-MSCs was assessed by increases in the alkaline phosphatase (ALP) activity. The amount of the osteogenic transcription factor Osterix and the alizarin red-stained bone nodule formation. ATP was measured with the luciferin-luciferase bioluminescence assay. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC RESULTS: The extracellular catabolism of ATP and UDP was faster in BM-MSCs from Pm women compared to younger females. The immunoreactivity against NTPDase3 increased 5.6-fold in BM-MSCs from Pm women vs. younger females. Selective inhibition or transient NTPDase3 gene silencing increased the extracellular accumulation of adenine and uracil nucleotides in cultured Pm BM-MSCs. Downregulation of NTPDase3 expression or activity rehabilitated the osteogenic commitment of Pm BM-MSCs measured as increases in ALP activity, Osterix protein cellular content and bone nodule formation; blockage of P2X7 and P2Y6 purinoceptors prevented this effect. CONCLUSIONS: Data suggest that NTPDase3 overexpression in BM-MSCs may be a clinical surrogate of the osteogenic differentiation impairment in Pm women. Thus, besides P2X7 and P2Y6 receptors activation, targeting NTPDase3 may represent a novel therapeutic strategy to increase bone mass and reduce the osteoporotic risk of fractures in Pm women.

P2X7-induced zeiosis promotes osteogenic differentiation and mineralization of postmenopausal bone marrow-derived mesenchymal stem cells.

Polymorphisms of the P2X7 receptor have been associated with increased risk of fractures in postmenopausal women. Although both osteoblasts and osteoclasts express P2X7 receptors, their function in osteogenesis remains controversial. Here, we investigated the role of the P2X7 receptor on osteogenic differentiation and mineralization of bone marrow mesenchymal stem cell (BMSC) cultures from postmenopausal women (age 71±3 yr, n=18). We focused on the mechanisms related to intracellular [Ca(2+)]i oscillations and plasma membrane-dynamics. ATP, and the P2X7 agonist BzATP (100 µM), increased [Ca(2+)]i in parallel to the formation of membrane pores permeable to TO-PRO-3 dye uptake. ATP and BzATP elicited reversible membrane blebs (zeiosis) in 38 ± 1 and 70 ± 1% of the cells, respectively. P2X7-induced zeiosis was Ca(2+) independent, but involved phospholipase C, protein kinase C, and Rho-kinase activation. BzATP (100 µM) progressively increased the expression of Runx-2 and Osterix transcription factors by 452 and 226% (at d 21), respectively, alkaline phosphatase activity by 88% (at d 28), and mineralization by 329% (at d 43) of BMSC cultures in a Rho-kinase-dependent manner. In summary, reversible plasma membrane zeiosis involving cytoskeleton rearrangements due to activation of the P2X7-Rho-kinase axis promotes osteogenic differentiation and mineralization of BMSCs, thus providing new therapeutic targets for postmenopausal bone loss.

Determinantes clínicos com influência no prognóstico da discectomia lombar: análise multifatorial / Clinical determinants' influence on lumbar discectomy prognosis: multi-factor analysis / Determinantes clínicos que influyen en el pronóstico de la disectomía lumbar: análisis multifactorial

A seleção e o agendamento temporal inadequados dos pacientes permanecem as causas mais importantes de insucesso da cirurgia discal lombar. OBJETIVO: Este trabalho pretende avaliar os resultados clínicos e os factores preditivos que influenciam o prognóstico dos pacientes submetidos à discectomia lombar através de um modelo analítico multifatorial. MÉTODOS: Apresentamos uma série prospectiva de 68 pacientes com hérnias discais lombares que foram submetidos à discectomia entre 2003 e 2007. O seguimento médio foi de 3,9±0,1 anos. Os resultados clínicos foram avaliados utilizando o escore JOA (Japonese Orthopaedic Association Assessment Scoring System) (JOA=29). RESULTADOS: Resultados pós-operatórios bom e excelente foram obtidos em 75 por cento dos casos. Pacientes do sexo masculino e a preponderância de sintomas subjectivos sobre sintomas clínicos estiveram associados com os melhores resultados clínicos e com o retorno mais precoce à actividade laboral. O tempo de espera pela cirurgia não teve influência no resultado final. A necessidade de cirurgia foi questionada na ausência de défices neurológicos significativos (JOA para resultados clínicos >3/6). Um valor JOA pré-operatório >15 (debilidade inicial baixa) está correlacionado com piores resultados clínicos pós-operatórios. CONCLUSÕES: O escore JOA é de fácil aplicação para clínicos e pacientes, uniformiza dados subjectivos e apresenta valor preditivo para os resultados dos pacientes submetidos à discectomia lombar.

Inappropriate patient selection and timing for lumbar disc surgery are still the the major causes of failures. OBJECTIVE: We evaluated the clinical outcome and predictive factors that influence the prognosis of patients submitted to lumbar discectomy using a multivariate analysis model. METHODS: We report a prospective series of 68 patients with herniated lumbar disc who underwent limited discectomy during 2003 to 2007. The average follow-up period was 3.9±0.1 years. Clinical findings were evaluated using Japonese Orthopaedic Association Assessment Scoring System (JOA=29). RESULTS: Excellent and good postoperative improvement rates were obtained in 75 percent of the patients. Male gender and preponderance of subjective symptoms over clinical signs were associated with better clinical outcomes and faster return to work. Waiting time for surgery had no consistent effect on the outcome. The need for immediate surgery was questionable in the absence of significant neurological disturbance (JOA for clinical signs >3/6). JOA scoring had predictive value for preoperative assessment for lumbar disc surgery, i.e. preoperative JOA >15 (low initial disability) correlated with poor surgery outcome. CONCLUSIONS: JOA scoring is easily performed for patients and clinicians, standardizes subjective data and presents high predictive value of patients' outcome after lumbar discectomy.

La selección y la agenda temporal inadecuada de los enfermos permanecen como las causas más importantes de la falta de éxito en la cirugía discal lumbar. OBJETIVO: este trabajo pretende evaluar los resultados clínicos y los factores predictivos que influyen en el pronóstico de los pacientes sometidos a la discectomía lumbar por medio de un modelo analítico multifatorial. MÉTODOS: se presenta una serie prospectiva de 68 pacientes con hernias discales lumbares, los cuales fueron sometidos a la discectomía entre 2003 y 2007. El seguimiento promedio fue de 3.9±0.1 años. Los resultados clínicos fueron evaluados utilizando el JOA (Japonese Orthopaedic Association Assessment Scoring System) score (JOA=29). RESULTADOS: los resultados postoperatorios buenos y excelentes fueron obtenidos en 75 por ciento de los pacientes. Enfermos del sexo masculino y la preponderancia de síntomas subjetivos sobre síntomas clínicos estuvieron asociados con los mejores resultados clínicos y con el regreso más precoz a la actividad laboral. El tiempo de espera para la cirugía no influyó en el resultado final. La necesidad de cirugía fue cuestionada en la ausencia de déficits neurológicos significativos (JOA para resultados clínicos >3/6). Un valor JOA preoperatorio > 15 (debilidad inicial baja) está correlacionado con los peores resultados clínicos postoperatorios. CONCLUSIONES: el JOA score se presenta de fácil aplicación para clínicos y enfermos, uniformiza datos subjetivos y presenta un valor predictivo para los resultados de los pacientes sometidos a la discectomía lumbar.