RESUMO
Sustained pressure overload and fibrosis of the right ventricle (RV) are the leading causes of mortality in pulmonary arterial hypertension (PAH). Although the role of adenosine in PAH has been attributed to the control of pulmonary vascular tone, cardiac reserve, and inflammatory processes, the involvement of the nucleoside in RV remodelling remains poorly understood. Conflicting results exist on targeting the low-affinity adenosine A2B receptor (A2BAR) for the treatment of PAH mostly because it displays dual roles in acute vs. chronic lung diseases. Herein, we investigated the role of the A2BAR in the viability/proliferation and collagen production by cardiac fibroblasts (CFs) isolated from RVs of rats with monocrotaline (MCT)-induced PAH. CFs from MCT-treated rats display higher cell viability/proliferation capacity and overexpress A2BAR compared to the cells from healthy littermates. The enzymatically stable adenosine analogue, 5'-N-ethylcarboxamidoadenosine (NECA, 1-30 µM), concentration-dependently increased growth, and type I collagen production by CFs originated from control and PAH rats, but its effects were more prominent in cells from rats with PAH. Blockage of the A2BAR with PSB603 (100 nM), but not of the A2AAR with SCH442416 (100 nM), attenuated the proliferative effect of NECA in CFs from PAH rats. The A2AAR agonist, CGS21680 (3 and 10 nM), was virtually devoid of effect. Overall, data suggest that adenosine signalling via A2BAR may contribute to RV overgrowth secondary to PAH. Therefore, blockage of the A2AAR may be a valuable therapeutic alternative to mitigate cardiac remodelling and prevent right heart failure in PAH patients.
Assuntos
Hipertensão Pulmonar , Hipertensão Arterial Pulmonar , Animais , Humanos , Ratos , Adenosina-5'-(N-etilcarboxamida) , Modelos Animais de Doenças , Fibroblastos/metabolismo , Hipertensão Pulmonar/tratamento farmacológico , Hipertensão Pulmonar/metabolismo , Receptor A2B de Adenosina/metabolismoRESUMO
Metabolic reprogramming is a central hub in tumor development and progression. Therefore, several efforts have been developed to find improved therapeutic approaches targeting cancer cell metabolism. Recently, we identified the 7α-acetoxy-6ß-benzoyloxy-12-O-benzoylroyleanone (Roy-Bz) as a PKCδ-selective activator with potent anti-proliferative activity in colon cancer by stimulating a PKCδ-dependent mitochondrial apoptotic pathway. Herein, we investigated whether the antitumor activity of Roy-Bz, in colon cancer, could be related to glucose metabolism interference. The results showed that Roy-Bz decreased the mitochondrial respiration in human colon HCT116 cancer cells, by reducing electron transfer chain complexes I/III. Consistently, this effect was associated with downregulation of the mitochondrial markers cytochrome c oxidase subunit 4 (COX4), voltage-dependent anion channel (VDAC) and mitochondrial import receptor subunit TOM20 homolog (TOM20), and upregulation of synthesis of cytochrome c oxidase 2 (SCO2). Roy-Bz also dropped glycolysis, decreasing the expression of critical glycolytic markers directly implicated in glucose metabolism such as glucose transporter 1 (GLUT1), hexokinase 2 (HK2) and monocarboxylate transporter 4 (MCT4), and increasing TP53-induced glycolysis and apoptosis regulator (TIGAR) protein levels. These results were further corroborated in tumor xenografts of colon cancer. Altogether, using a PKCδ-selective activator, this work evidenced a potential dual role of PKCδ in tumor cell metabolism, resulting from the inhibition of both mitochondrial respiration and glycolysis. Additionally, it reinforces the antitumor therapeutic potential of Roy-Bz in colon cancer by targeting glucose metabolism.
Assuntos
Neoplasias do Colo , Complexo IV da Cadeia de Transporte de Elétrons , Humanos , Linhagem Celular Tumoral , Neoplasias do Colo/patologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Glucose/metabolismo , Glicólise , RespiraçãoRESUMO
Nucleotides released during heart injury affect myocardium electrophysiology and remodeling through P2 purinoceptors activation in cardiac myofibroblasts. ATP and UTP endorse [Ca2+ ]i accumulation and growth of DDR-2/α-SMA-expressing myofibroblasts from adult rat ventricles via P2Y4 and P2Y2 receptors activation, respectively. Ventricular myofibroblasts also express ADP-sensitive P2Y1 , P2Y12 , and P2Y13 receptors as demonstrated by immunofluorescence confocal microscopy and western blot analysis, but little information exists on ADP effects in these cells. ADP (0.003-3 mM) and its stable analogue, ADPßS (100 µM), caused fast [Ca2+ ]i transients originated from thapsigargin-sensitive internal stores, which partially declined to a plateau sustained by capacitative Ca2+ entry through transient receptor potential (TRP) channels inhibited by 2-APB (50 µM) and flufenamic acid (100 µM). Hydrophobic interactions between Gq/11 -coupled P2Y purinoceptors and TRP channels were suggested by prevention of the ADP-induced [Ca2+ ]i plateau following PIP2 depletion with LiCl (10 mM) and cholesterol removal from lipid rafts with methyl-ß-cyclodextrin (2 mM). ADP [Ca2+ ]i transients were insensitive to P2Y1 , P2Y12 , and P2Y13 receptor antagonists, MRS2179 (10µM), AR-C66096 (0.1 µM), and MRS2211 (10µM), respectively, but were attenuated by suramin and reactive blue-2 (100 µM) which also blocked P2Y4 receptors activation by UTP. Cardiac myofibroblasts growth and type I collagen production were favored upon activation of MRS2179-sensitive P2Y1 receptors with ADP or ADPßS (30 µM). In conclusion, ADP exerts a dual role on ventricular myofibroblasts: [Ca2+ ]i transients are mediated by fast-desensitizing P2Y4 receptors, whereas the pro-fibrotic effect of ADP involves the P2Y1 receptor activation. Data also show that ADP-induced capacitative Ca2+ influx depends on phospholipase C-linked TRP channels opening in lipid raft microdomains. J. Cell. Physiol. 232: 1511-1526, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Difosfato de Adenosina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Ventrículos do Coração/citologia , Ativação do Canal Iônico/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Miofibroblastos/metabolismo , Canais de Potencial de Receptor Transitório/metabolismo , Fosfolipases Tipo C/metabolismo , Difosfato de Adenosina/análogos & derivados , Envelhecimento , Animais , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Interações Hidrofóbicas e Hidrofílicas , Masculino , Miofibroblastos/efeitos dos fármacos , Proteína Quinase C/metabolismo , Ratos Wistar , Receptores Purinérgicos P2Y/metabolismo , Trocador de Sódio e Cálcio/metabolismoRESUMO
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.
Assuntos
Osso e Ossos/citologia , Calcificação Fisiológica/fisiologia , Diferenciação Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Pós-Menopausa , Receptores Purinérgicos P2X7/fisiologia , Idoso , Cálcio/metabolismo , Ativação Enzimática , Feminino , Humanos , Células-Tronco Mesenquimais/enzimologia , Proteína Quinase C/metabolismo , Fosfolipases Tipo C/metabolismo , Quinases Associadas a rho/metabolismoRESUMO
Changes in the regulation of connective tissue ATP-mediated mechano-transduction and remodeling may be an important link to the pathogenesis of chronic pain. It has been demonstrated that mast cell-derived histamine plays an important role in painful fibrotic diseases. Here we analyzed the involvement of ATP in the response of human subcutaneous fibroblasts to histamine. Acute histamine application caused a rise in intracellular Ca(2+) ([Ca(2+)]i) and ATP release from human subcutaneous fibroblasts via H1 receptor activation. Histamine-induced [Ca(2+)]i rise was partially attenuated by apyrase, an enzyme that inactivates extracellular ATP, and by blocking P2 purinoceptors with pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) tetrasodium salt and reactive blue 2. [Ca(2+)]i accumulation caused by histamine was also reduced upon blocking pannexin-1 hemichannels with (10)Panx, probenecid, or carbenoxolone but not when connexin hemichannels were inhibited with mefloquine or 2-octanol. Brefeldin A, an inhibitor of vesicular exocytosis, also did not block histamine-induced [Ca(2+)]i mobilization. Prolonged exposure of human subcutaneous fibroblast cultures to histamine favored cell growth and type I collagen synthesis via the activation of H1 receptor. This effect was mimicked by ATP and its metabolite, ADP, whereas the selective P2Y1 receptor antagonist, MRS2179, partially attenuated histamine-induced cell growth and type I collagen production. Expression of pannexin-1 and ADP-sensitive P2Y1 receptor on human subcutaneous fibroblasts was confirmed by immunofluorescence confocal microscopy and Western blot analysis. In conclusion, histamine induces ATP release from human subcutaneous fibroblasts, via pannexin-1 hemichannels, leading to [Ca(2+)]i mobilization and cell growth through the cooperation of H1 and P2 (probably P2Y1) receptors.
Assuntos
Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Conexinas/metabolismo , Fibroblastos/metabolismo , Agonistas dos Receptores Histamínicos/farmacologia , Histamina/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Difosfato de Adenosina/análogos & derivados , Difosfato de Adenosina/metabolismo , Difosfato de Adenosina/farmacologia , Antibacterianos/farmacologia , Antimaláricos/farmacologia , Brefeldina A/farmacologia , Células Cultivadas , Colágeno Tipo I/biossíntese , Conexinas/antagonistas & inibidores , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Feminino , Fibroblastos/citologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Histamina/metabolismo , Agonistas dos Receptores Histamínicos/metabolismo , Humanos , Masculino , Mastócitos/citologia , Mastócitos/metabolismo , Mefloquina/farmacologia , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/antagonistas & inibidores , Octanóis/farmacologia , Antagonistas do Receptor Purinérgico P2Y/farmacologia , Receptores Histamínicos H1/metabolismo , Receptores Purinérgicos P2Y1/metabolismoRESUMO
BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model. METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation. RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model. CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.
Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais , Osteogênese , Pós-Menopausa , Feminino , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Humanos , Osteogênese/efeitos dos fármacos , Animais , Idoso , Ratos , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Transplante de Células-Tronco Mesenquimais/métodos , Fator de Transcrição Sp7/metabolismo , Fator de Transcrição Sp7/genética , Células Cultivadas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Ratos WistarRESUMO
BACKGROUND: Chronic musculoskeletal pain involves connective tissue remodeling triggered by inflammatory mediators, such as bradykinin. Fibroblast cells signaling involve changes in intracellular Ca2+ ([Ca2+]i). ATP has been related to connective tissue mechanotransduction, remodeling and chronic inflammatory pain, via P2 purinoceptors activation. Here, we investigated the involvement of ATP in bradykinin-induced Ca2+ signals in human subcutaneous fibroblasts. RESULTS: Bradykinin, via B2 receptors, caused an abrupt rise in [Ca2+]i to a peak that declined to a plateau, which concentration remained constant until washout. The plateau phase was absent in Ca2+-free medium; [Ca2+]i signal was substantially reduced after depleting intracellular Ca2+ stores with thapsigargin. Extracellular ATP inactivation with apyrase decreased the [Ca2+]i plateau. Human subcutaneous fibroblasts respond to bradykinin by releasing ATP via connexin and pannexin hemichannels, since blockade of connexins, with 2-octanol or carbenoxolone, and pannexin-1, with 10Panx, attenuated bradykinin-induced [Ca2+]i plateau, whereas inhibitors of vesicular exocytosis, such as brefeldin A and bafilomycin A1, were inactive. The kinetics of extracellular ATP catabolism favors ADP accumulation in human fibroblast cultures. Inhibition of ectonucleotidase activity and, thus, ADP formation from released ATP with POM-1 or by Mg2+ removal from media reduced bradykinin-induced [Ca2+]i plateau. Selective blockade of the ADP-sensitive P2Y12 receptor with AR-C66096 attenuated bradykinin [Ca2+]i plateau, whereas the P2Y1 and P2Y13 receptor antagonists, respectively MRS 2179 and MRS 2211, were inactive. Human fibroblasts exhibited immunoreactivity against connexin-43, pannexin-1 and P2Y12 receptor. CONCLUSIONS: Bradykinin induces ATP release from human subcutaneous fibroblasts via connexin and pannexin-1-containing hemichannels leading to [Ca2+]i mobilization through the cooperation of B2 and P2Y12 receptors.
Assuntos
Trifosfato de Adenosina/metabolismo , Bradicinina/metabolismo , Sinalização do Cálcio/fisiologia , Fibroblastos/metabolismo , Receptor B2 da Bradicinina/metabolismo , Receptores Purinérgicos P2Y12/metabolismo , Difosfato de Adenosina/metabolismo , Células Cultivadas , Conexina 43/metabolismo , Conexinas/metabolismo , Humanos , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/metabolismoRESUMO
Radiation therapy (RT) is a clinical modality treatment that uses ionizing radiation in the approach of malignant tumors, (and occasionally benign diseases). Since its inception, the goal of RT has been to cure cancer without excessive side effects. The most important factors affecting the results of RT are the tumor histology, its location and regional extent, the anatomic area of involvement and the geometric accuracy with which a calculated radiation dose is delivered. Radiotherapy is one of the basic treatment modalities in thoracic malignancies and is used across all histology types and stages. Technical developments of radiotherapy have further strengthened and redefined its place in the management of lung cancer. High precision intensity-modulated radiation therapy, volumetric modulated arc therapy, and stereotactic body radiation therapy (SBRT), coupled with management of tumor motion and on-board imaging, increased efficacy and markedly decreased treatment-related toxicity. With this brief review, the authors pretend to present basic concepts and recent techniques advances in the use of radiation therapy for thoracic malignancies.
Assuntos
Neoplasias Pulmonares , Radiocirurgia , Radioterapia de Intensidade Modulada , Humanos , Neoplasias Pulmonares/radioterapia , Radioterapia de Intensidade Modulada/efeitos adversos , Radiocirurgia/métodos , Dosagem Radioterapêutica , Planejamento da Radioterapia Assistida por Computador/métodosRESUMO
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.
Assuntos
Células-Tronco Mesenquimais , Osteogênese , Humanos , Feminino , Idoso , Adolescente , Adulto Jovem , Adulto , Pós-Menopausa , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Nucleotídeos de Uracila/metabolismo , Nucleotídeos de Uracila/farmacologia , Difosfato de Uridina/metabolismo , Difosfato de Uridina/farmacologia , Trifosfato de Adenosina/metabolismo , Células da Medula Óssea , Células CultivadasRESUMO
AIMS: Disorganization of the subcutaneous tissue due to inflammation and fibrosis is a common feature in patients with myofascial pain. Dermal accumulation of adenosine favours collagen production by human subcutaneous fibroblasts (HSCF) via A2A receptors (A2AR) activation. Adenosine mimics the fibrogenic effect of inflammatory mediators (e.g. histamine, bradykinin), which promote ATP release from HSCF via plasma-membrane-bound pannexin-1 (Panx1) and/or connexin-43 (Cx43) channels, but this mechanism has never been implicated in A2AR actions. MATERIALS AND METHODS: A2AR-mediated effects on Panx1 and Cx43 protein amounts were evaluated in primary cultures of HSCF by confocal microscopy and Western blot analysis. Functional repercussions in collagen production, intracellular [Ca2+]i oscillations and ATP release were also evaluated. KEY FINDINGS: NECA and CGS21680, two enzymatically-stable A2AR agonists, increased Panx1, but reduced Cx43, protein density in HSCF. This effect was accompanied by increases in ATP release and collagen III production by HSCF. The involvement of the A2AR was confirmed by blockage with the selective A2AR antagonist, SCH442416. Inhibition of Panx1 channels by probenecid and the Panx1 mimetic inhibitory peptide, 10Panx, also decreased ATP release and collagen production by HSCF under similar conditions. Superfluous ATP release by HSCF exposed to A2AR agonists overexpressing Panx1 channels contributes to keeping high [Ca2+]i levels when the cells were exposed to histamine. SIGNIFICANCE: Adenosine A2AR-induced Panx1 overexpression was shown here for the first time in HSCF; this feature indirectly implicates ATP release in the fibrogenic vicious cycle operated by adenosine accumulating in subcutaneous tissue fibrosis and myofascial pain associated to dermal inflammation.
Assuntos
Conexina 43 , Conexinas , Proteínas do Tecido Nervoso , Receptor A2A de Adenosina , Humanos , Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Colágeno/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Fibroblastos/metabolismo , Fibrose , Histamina/metabolismo , Inflamação/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Dor/metabolismo , Receptor A2A de Adenosina/metabolismo , Tela Subcutânea/metabolismoRESUMO
Human subcutaneous fibroblasts (HSCF) challenged with inflammatory mediators release huge amounts of ATP, which rapidly generates adenosine. Given the nucleoside's putative relevance in wound healing, dermal fibrosis, and myofascial pain, we investigated the role of its precursor, AMP, and of its metabolite, inosine, in HSCF cells growth and collagen production. AMP (30 µM) was rapidly (t½ 3 ± 1 min) dephosphorylated into adenosine by CD73/ecto-5'-nucleotidase. Adenosine accumulation (t½ 158 ± 17 min) in the extracellular fluid reflected very low cellular adenosine deaminase (ADA) activity. HSCF stained positively against A2A and A3 receptors but were A1 and A2B negative. AMP and the A2A receptor agonist, CGS21680C, increased collagen production without affecting cells growth. The A2A receptor antagonist, SCH442416, prevented the effects of AMP and CGS21680C. Inosine and the A3 receptor agonist, 2Cl-IB-MECA, decreased HSCF growth and collagen production in a MRS1191-sensitive manner, implicating the A3 receptor in the anti-proliferative action of inosine. Incubation with ADA reproduced the inosine effect. In conclusion, adenosine originated from extracellular ATP hydrolysis favors normal collagen production by HSCF via A2A receptors. Inhibition of unpredicted inosine formation by third party ADA cell providers (e.g., inflammatory cells) may be a novel therapeutic target to prevent inappropriate dermal remodeling via A3 receptors activation.
Assuntos
5'-Nucleotidase/efeitos dos fármacos , Adenosina Desaminase/metabolismo , Adenosina/análogos & derivados , Fibroblastos/efeitos dos fármacos , Inosina/farmacologia , 5'-Nucleotidase/metabolismo , AMP Cíclico/metabolismo , Fibroblastos/metabolismo , Proteínas Ligadas por GPI/efeitos dos fármacos , Humanos , Inosina/metabolismoRESUMO
Obesity is a worldwide health problem which have reached pandemic proportions, now also including low and middle-income countries. Excessive or abnormal fat deposition in the abdomen especially in the visceral compartment is tightly associated with a high metabolic risk for arterial hypertension, type II diabetes, cardiovascular diseases, musculoskeletal disorders (especially articular degeneration) and some cancers. Contrariwise, accumulation of fat in the subcutaneous compartment has been associated with a neutral metabolic impact, favoring a lower risk of insulin resistance. Obesity results more often from an avoidable imbalance between food consumption and energy expenditure. There are several recommended strategies for dealing with obesity, including pharmacological therapies, but their success remains incomplete and may not compensate the associated adverse effects. Purinergic signaling operated by ATP and its metabolite, adenosine, has attracted increasing attention in obesity. The extracellular levels of purines often reflect the energy status of a given cell population. Adenine nucleotides and nucleosides fine tuning control adipogenesis and mature adipocytes function via the activation of P2 and P1 purinoceptors, respectively. These features make the purinergic signaling cascade a putative target for therapeutic intervention in obesity and related metabolic syndromes. There are, however, gaps in our knowledge regarding the role of purines in adipocyte precursors differentiation and mature adipocytes functions, as well as their impact among distinct adipose tissue deposits (e.g. white vs. brown, visceral vs. subcutaneous), which warrants further investigations before translation to clinical trials can be made.
Assuntos
Adipogenia/fisiologia , Obesidade/metabolismo , Purinas/metabolismo , Receptores Purinérgicos P1/metabolismo , Receptores Purinérgicos/metabolismo , Transdução de Sinais/fisiologia , Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Animais , Humanos , Obesidade/patologiaRESUMO
During myocardial ischemia and reperfusion both purines and pyrimidines are released into the extracellular milieu, thus creating a signaling wave that propagates to neighboring cells via membrane-bound P2 purinoceptors activation. Cardiac fibroblasts (CF) are important players in heart remodeling, electrophysiological changes and hemodynamic alterations following myocardial infarction. Here, we investigated the role UTP on calcium signaling and proliferation of CF cultured from ventricles of adult rats. Co-expression of discoidin domain receptor 2 and α-smooth muscle actin indicate that cultured CF are activated myofibroblasts. Intracellular calcium ([Ca(2+)]i) signals were monitored in cells loaded with Fluo-4 NW. CF proliferation was evaluated by the MTT assay. UTP and the selective P2Y4 agonist, MRS4062, caused a fast desensitizing [Ca(2+)]i rise originated from thapsigargin-sensitive internal stores, which partially declined to a plateau providing the existence of Ca(2+) in the extracellular fluid. The biphasic [Ca(2+)]i response to UTP was attenuated respectively by P2Y4 blockers, like reactive blue-2 and suramin, and by the P2Y11 antagonist, NF340. UTP and the P2Y2 receptor agonist MRS2768 increased, whereas the selective P2Y11 agonist NF546 decreased, CF growth; MRS4062 was ineffective. Blockage of the P2Y11 receptor or its coupling to adenylate cyclase boosted UTP-induced CF proliferation. Confocal microscopy and Western blot analysis confirmed the presence of P2Y2, P2Y4 and P2Y11 receptors. Data indicate that besides P2Y4 and P2Y2 receptors which are responsible for UTP-induced [Ca(2+)]i transients and growth of CF, respectively, synchronous activation of the previously unrecognized P2Y11 receptor may represent an important target for anti-fibrotic intervention in cardiac remodeling.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Fibroblastos/metabolismo , Miocárdio/citologia , Receptores Purinérgicos P2/metabolismo , Uridina Trifosfato/farmacologia , Animais , Cálcio/análise , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Masculino , Ratos , Ratos Wistar , Relação Estrutura-AtividadeRESUMO
We attempted to prepare chitosan-silicate hybrid for use in a medical application and evaluated the physico-chemical properties and osteocompatibility of the hybrids as a function of gamma-glycidoxypropyltrimethoxysilane (GPTMS) concentration. Chitosan-silicate hybrids were synthesized using GPTMS as the reagent for cross-linking of the chitosan chains. Fourier transform infrared spectroscopy, (29)Si CP-MAS NMR spectroscopy and the ninhydrin assay were used to analyze the structures of the hybrids, and stress-strain curves were recorded to estimate their Young's modulus. The swelling ability, contact angle and cytocompatibility of the hybrids were investigated as a function of the GPTMS concentration. A certain fraction of GPTMS in each hybrid was linked at the epoxy group to the amino group of chitosan, which was associated with the change in the methoxysilane group of GPTMS due to hybridization. The cross-linking density was around 80% regardless of the volume of GPTMS. As the content of GPTMS increased, the water uptake decreased and the hydrophilicity of the hybrids increased except when the content exceeded amolar ratio of 1.5, when it caused a decrease. The values of the mechanical parameters assessed indicated that significant stiffening of the hybrids was obtained by the addition of GPTMS. The adhesion and proliferation of the MG63 osteoblast cells cultured on the chitosan-GPTMS hybrid surface were improved compared to those on the chitosan membrane, regardless of the GPTMS concentration. Moreover, human bone marrow osteoblast cells proliferated on the chitosan-GPTMS hybrid surface and formed a fibrillar extracellular matrix with numerous calcium phosphate globular structures, both in the presence and in the absence of dexamethasone. Therefore, the chitosan-GPTMS hybrids are promising candidates for basic materials that can promote bone regeneration because of their controllable composition (chitosan/GPTMS ratio).
Assuntos
Quitosana/química , Silanos/química , Siloxanas/química , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Reagentes de Ligações Cruzadas/química , Humanos , Espectroscopia de Ressonância Magnética , Microscopia Eletrônica de Varredura , Osteoblastos/metabolismo , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de Fourier , Engenharia Tecidual/métodosRESUMO
Human gingival (HG), periodontal ligament (HPL) and alveolar bone (HAB) cells (first subculture) were cultured (10(4) cells/cm2) for 35 days in alpha-Minimal Essential Medium supplemented with 10% fetal bovine serum in the presence of (i) ascorbic acid (AA, 50 microg/mL), (ii) AA + beta-glycerophosphate (betaGP, 10 mM) and (iii) AA + betaGP + dexamethasone (Dex, 10 nM). Cultures were assessed for cell attachment and spreading, cell proliferation, alkaline phosphatase (ALP) and acid phosphatase (ACP) activities and matrix mineralization. HG cell cultures presented a high proliferation rate, a low ability to synthesize ALP and ACP and the formation of a non-mineralized extracellular matrix, regardless the experimental situation. HPL cell cultures were very sensitive to the culture conditions and showed a high proliferation rate, synthesis of moderate levels of ALP and ACP and a modest matrix mineralization in the presence of AA + betaGP + Dex. HAB cell cultures presented a growth rate lower than that of HG and HPL cells, a high ALP activity and comparatively low levels of ACP, and the ready formation of a heavy mineralized matrix in the presence of betaGP. In the three periodontal cell cultures, Dex enhanced cell proliferation and expression of osteoblastic markers. Results showed that betaGP and Dex allowed the modulation of the cell proliferation/differentiation behavior within the proposed physiological and regenerative capabilities of these periodontal cells.
Assuntos
Osso e Ossos/citologia , Técnicas de Cultura de Células/métodos , Gengiva/citologia , Ligamento Periodontal/citologia , Fosfatase Ácida/metabolismo , Fosfatase Alcalina/metabolismo , Fosfatos de Cálcio/metabolismo , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Humanos , Fatores de TempoRESUMO
OBJETIVOS: O objetivo do presente estudo é avaliar a fidedignidade de um instrumento destinado a mensurar a satisfação de doadores de sangue em um centro de hematologia e transfusão. SUJEITOS E MÉTODOS: Foi desenvolvido um questionário objetivando mensurar a satisfação dos doadores de sangue do Hemocentro/Ribeirão Preto, onde os mesmos classificaram 18 afirmativas em uma escala de Likert de 4 itens, variando de "totalmente errado" a "totalmente certo". Uma amostra de 407 doadores foi obtida sistematicamente, onde, a cada quatro pessoas que seqüencialmente compareceram para doação, o quarto era convidado a preencher o questionário. Uma amostra final de 407 doadores foi utilizada para a análise da fidedignidade do questionário e análises preliminares. A análise de correspondência (AC) foi utilizada para avaliação do grau de satisfação dos doadores para cada item. RESULTADOS: Os participantes tinham em média 31,2 anos (desvio padrão de 9,13 e amplitude de 18 a 67 anos). Foi encontrado um coeficiente alfa de Cronbach de 0,97, indicando uma consistência interna muito alta do questionário. A AC indicou que os itens com maior insatisfação são o acesso ao Hemocentro e o tempo gasto na doação. Itens de maior satisfação referem-se à confiança no serviço, funcionários e atendimento. A AC identificou que, de modo geral, as pessoas que estão doando sangue pela primeira vez tendem a uma menor satisfação. CONCLUSÃO: A alta consistência interna do questionário indica que os itens abordados têm alto poder de mensurar um mesmo objeto, mas ainda há a necessidade de estudar a validação do instrumento para avaliar se tal objeto é necessariamente a satisfação do doador.
Assuntos
Bancos de Sangue , Doadores de Sangue , Inquéritos e Questionários , Comportamento do ConsumidorRESUMO
Muito haveria que dizer acerca da História da Medicina e da descoberta dos Rx e da Radioatividade, o que näo é âmbito desta nossa breve revisäo histórica. Pretendemos apenas prestar uma pequena homenagem a todos aqueles que, pelo seu esforço e dedicaçäo, contribuíram para que nós hoje possamos exercer a Especialidade Médica que escolhemos. Todos os nomes que citamos fizeram história, bem como tantos outros que ficaram por citar.
Assuntos
Humanos , Masculino , Feminino , História da Medicina , Radioterapia/história , Raios XRESUMO
O leiomiossarcoma primário do pulmÝo é considerado uma entidade extremamente rara. Os achados radiológicos bem como a sua apresentaçÝo clínica nÝo sÝo patognomônicos. Ao contrário do carcinoma brônquico mantém-se confinado ao pulmÝo por muito mais tempo. A metastizaçÝo ocorre tardiamente e em menos de 1/3 dos casos. O tratamento de eleiçÝo é a cirurgia. Os autores apresentam um caso de uma doente de 50 anos de idade com o diagnóstico de leiomiossarcoma do pulmÝo de alto grau de malignidade, seu prognóstico, tratamento e evoluçÝo.