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1.
Medicine (Baltimore) ; 99(7): e17763, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32049775

RESUMO

Unstable distal metaphyseal and dia-metaphyseal fractures of the radius may have treated with a variety of operative techniques, Kirschner wires (K-wires), dorsally inserted titanium elastic stable intramedullary nailing (DESIN), and short titanium elastic stable intramedullary nailing (SESIN) in children.The aim of this study was to evaluate the differences in clinical and radiographic outcomes between these methods.Between January 2009 and December 2017 196 children were treated for forearm fractures in the distal third of the distal radius. Gender of the patients, different types of surgical techniques, number of postoperative X-rays, date of metal removal and degree of axis deviation after the metal removal were studied. Distance of the fracture line from the radiocarpal surface, the width of the distal epiphysis of the radius, and the cumulative width of the distal epiphysis of the ulna and radius were analyzed.Out of the 196 children, stabilization of the fracture was achieved by K-wire in 139, by DESIN in 44, and by SESIN in 13 patients. The average time of metal removal was significantly shorter (3.8 months), following stabilization with K-wire. In children treated with K-wire, axial deviation of <5° was seen in 118 patients, 5° to 10° deviation in 15 patients, while deviation was above 10° in 6 children. In the DESIN group, <5° axial deviation was found in 37 patients and 5° to 10° in seven patients. In all 13 children treated with SESIN, axial deviation was measured to be <5°. The fracture distance from the radiocarpal surface was on average 23.7 and 45.6 mm in the children treated with K-wire and DESIN, respectively.Fracture distance from the radiocarpal surface might determine the type of surgical technique required. If the distance of the fracture line is less than the width of the distal radius, osteosynthesis with a K-wire is recommended, while if the distance of the fracture is more than the cumulative width of the radius and the ulna, then DESIN may provide better results. The use of SESIN may be indicated when the area of the growth plate is injured.

2.
J Mol Neurosci ; 2019 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-31808034

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally secreted signaling peptide and has important regulatory roles in the differentiation of the central nervous system and its absence results in disorders in femur development. PACAP has an important function in prevention of oxidative stress or mechanical stress in chondrogenesis but little is known about its function in bone regeneration. A new callus formation model was set to investigate its role in bone remodeling. Fracturing was 5 mm distal from the proximal articular surface of the tibia and the depth was 0.5 mm. Reproducibility of callus formation was investigated with CT 3, 7, and 21 days after the operation. Absence of PACAP did not alter the alkaline phosphatase (ALP) activation in PACAP KO healing process. In developing callus, the expression of collagen type I increased in wild-type (WT) and PACAP KO mice decreased to the end of healing process. Expression of the elements of BMP signaling was disturbed in the callus formation of PACAP KO mice, as bone morphogenic protein 4 (BMP4) and 6 showed an early reduction in bone regeneration. However, elevated Smad1 expression was demonstrated in PACAP KO mice. Our results indicate that PACAP KO mice show various signs of disturbed bone healing and suggest PACAP compensatory and fine tuning effects in proper bone regeneration.

3.
Cell Commun Signal ; 17(1): 166, 2019 Dec 16.
Artigo em Inglês | MEDLINE | ID: mdl-31842918

RESUMO

BACKGROUND: In vitro chondrogenesis depends on the concerted action of numerous signalling pathways, many of which are sensitive to the changes of intracellular Ca2+ concentration. N-methyl-D-aspartate (NMDA) glutamate receptor is a cation channel with high permeability for Ca2+. Whilst there is now accumulating evidence for the expression and function of NMDA receptors in non-neural tissues including mature cartilage and bone, the contribution of glutamate signalling to the regulation of chondrogenesis is yet to be elucidated. METHODS: We studied the role of glutamatergic signalling during the course of in vitro chondrogenesis in high density chondrifying cell cultures using single cell fluorescent calcium imaging, patch clamp, transient gene silencing, and western blotting. RESULTS: Here we show that key components of the glutamatergic signalling pathways are functional during in vitro chondrogenesis in a primary chicken chondrogenic model system. We also present the full glutamate receptor subunit mRNA and protein expression profile of these cultures. This is the first study to report that NMDA-mediated signalling may act as a key factor in embryonic limb bud-derived chondrogenic cultures as it evokes intracellular Ca2+ transients, which are abolished by the GluN2B subunit-specific inhibitor ifenprodil. The function of NMDARs is essential for chondrogenesis as their functional knock-down using either ifenprodil or GRIN1 siRNA temporarily blocks the differentiation of chondroprogenitor cells. Cartilage formation was fully restored with the re-expression of the GluN1 protein. CONCLUSIONS: We propose a key role for NMDARs during the transition of chondroprogenitor cells to cartilage matrix-producing chondroblasts.

4.
Geroscience ; 41(6): 775-793, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655957

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is an evolutionarly conserved neuropeptide which is produced by various neuronal and non-neuronal cells, including cartilage and bone cells. PACAP has trophic functions in tissue development, and it also plays a role in cellular and tissue aging. PACAP takes part in the regulation of chondrogenesis, which prevents insufficient cartilage formation caused by oxidative and mechanical stress. PACAP knockout (KO) mice have been shown to display early aging signs affecting several organs. In the present work, we investigated articular cartilage of knee joints in young and aged wild-type (WT) and PACAP KO mice. A significant increase in the thickness of articular cartilage was detected in aged PACAP gene-deficient mice. Amongst PACAP receptors, dominantly PAC1 receptor was expressed in WT knee joints and a remarkable decrease was found in aged PACAP KO mice. Expression of PKA-regulated transcription factors, Sox5, Sox9 and CREB, decreased both in young and aged gene deficient mice, while Sox6, collagen type II and aggrecan expressions were elevated in young but were reduced in aged PACAP KO animals. Increased expression of hyaluronan (HA) synthases and HA-binding proteins was detected parallel with an elevated presence of HA in aged PACAP KO mice. Expression of bone related collagens (I and X) was augmented in young and aged animals. These results suggest that loss of PACAP signaling results in dysregulation of cartilage matrix composition and may transform articular cartilage in a way that it becomes more prone to degenerate.

5.
PLoS One ; 14(1): e0211433, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30682157

RESUMO

BACKGROUND: PACAP and VIP are closely related neuropeptides with wide distribution and potent effect in the vasculature. We previously reported vasomotor activity in peripheral vasculature of male wild type (WT) and PACAP-deficient (KO) mice. However, female vascular responses are still unexplored. We hypothesized that PACAP-like activity is maintained in female PACAP KO mice and the mechanism through which it is regulated differs from that of male PACAP KO animals. METHODS: We investigated the vasomotor effects of VIP and PACAP isoforms and their selective blockers in WT and PACAP KO female mice in carotid and femoral arteries. The expression and level of different PACAP receptors in the vessels were measured by RT-PCR and Western blot. RESULTS: In both carotid and femoral arteries of WT mice, PACAP1-38, PACAP1-27 or VIP induced relaxation, without pronounced differences between them. Reduced relaxation was recorded only in the carotid arteries of KO mice as compared to their WT controls. The specific VPAC1R antagonist completely blocked the PACAP/VIP-induced relaxation in both arteries of all mice, while PAC1R antagonist affected relaxation only in their femoral arteries. CONCLUSION: In female WT mice, VPAC1 receptors appear to play a dominant role in PACAP-induced vasorelaxation both in carotid and in femoral arteries. In the PACAP KO group PAC1R activation exerts vasorelaxation in the femoral arteries but in carotid arteries there is no significant effect of the activation of this receptor. In the background of this regional difference, decreased PAC1R and increased VPAC1R availability in the carotid arteries was found.


Assuntos
Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Vasodilatação , Animais , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/fisiologia , Feminino , Artéria Femoral/efeitos dos fármacos , Artéria Femoral/fisiologia , Proteínas de Insetos/farmacologia , Camundongos , Camundongos Knockout , Nitroprussiato/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/antagonistas & inibidores , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatação/efeitos dos fármacos
6.
Int J Mol Sci ; 20(1)2019 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-30621194

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide also secreted by non-neural cells, including chondrocytes. PACAP signaling is involved in the regulation of chondrogenesis, but little is known about its connection to matrix turnover during cartilage formation and under cellular stress in developing cartilage. We found that the expression and activity of hyaluronidases (Hyals), matrix metalloproteinases (MMP), and aggrecanase were permanent during the course of chondrogenesis in primary chicken micromass cell cultures, although protein levels changed daily, along with moderate and relatively constant enzymatic activity. Next, we investigated whether PACAP influences matrix destructing enzyme activity during oxidative and mechanical stress in chondrogenic cells. Exogenous PACAP lowered Hyals and aggrecanase expression and activity during cellular stress. Expression and activation of the majority of cartilage matrix specific MMPs such as MMP1, MMP7, MMP8, and MMP13, were also decreased by PACAP addition upon oxidative and mechanical stress, while the activity of MMP9 seemed not to be influenced by the neuropeptide. These results suggest that application of PACAP can help to preserve the integrity of the newly synthetized cartilage matrix via signaling mechanisms, which ultimately inhibit the activity of matrix destroying enzymes under cellular stress. It implies the prospect that application of PACAP can ameliorate articular cartilage destruction in joint diseases.


Assuntos
Proteínas Reguladoras de Apoptose/farmacologia , Condrócitos/efeitos dos fármacos , Estresse Oxidativo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Estresse Mecânico , Animais , Proteínas Reguladoras de Apoptose/administração & dosagem , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Técnicas de Cultura de Células , Embrião de Galinha , Condrócitos/metabolismo , Endopeptidases/metabolismo , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Hialuronoglucosaminidase/metabolismo , Peróxido de Hidrogênio/farmacologia , Metaloproteinases da Matriz/metabolismo , Oxidantes/farmacologia
7.
J Mol Neurosci ; 68(3): 408-419, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30443839

RESUMO

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide that exerts general cytoprotective effects, including protection in different kidney disorders. The aim of our study was to investigate the ischemia/reperfusion-induced kidney injury of male and female rats to confirm the protective effects of PACAP in the kidney and to reveal possible gender differences.Male and female Wistar rats underwent unilateral renal artery clamping followed by 24-h, 48-h, or 14-day reperfusion. PACAP was administered intravenously before arterial clamping in half of the rats. Tubular damage, cytokine expression pattern, oxidative stress marker, antioxidative status and signaling pathways were evaluated using histology, immunohistology, cytokine array, PCR, and Western blot. Tubular damage was significantly less severe in the PACAP-treated male and female rats compared to controls. Results of female animals were significantly better in both treated and untreated groups. Cytokine expression, oxidative stress marker and antioxidative status confirmed the histological results. We also revealed that PACAP counteracted the decreased PKA phosphorylation, influenced the expression of BMP2 and BMP4, and increased the expression of the protein Smad1.We conclude that PACAP is protective in ischemia/reperfusion-induced kidney injury in both sexes, but females had markedly less pronounced injury after ischemia/reperfusion, possibly also involving further protective factors, the investigation of which could have future therapeutic value in treating ischemic kidney injuries.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Anti-Inflamatórios/uso terapêutico , Antioxidantes/uso terapêutico , Rim/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/uso terapêutico , Traumatismo por Reperfusão/tratamento farmacológico , Lesão Renal Aguda/etiologia , Animais , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Citocinas/sangue , Feminino , Rim/irrigação sanguínea , Rim/metabolismo , Masculino , Estresse Oxidativo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Ratos , Ratos Wistar , Traumatismo por Reperfusão/complicações , Fatores Sexuais , Proteína Smad1/genética , Proteína Smad1/metabolismo
8.
J Neuroinflammation ; 15(1): 335, 2018 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-30509328

RESUMO

OBJECTIVE: The regulatory role of capsaicin-sensitive peptidergic sensory nerves has been shown in acute inflammation, but little is known about their involvement in T/B-cell driven autoimmune arthritis. This study integratively characterized the function of these nerve endings in the proteoglycan-induced chronic arthritis (PGIA), a translational model of rheumatoid arthritis. METHODS: Peptidergic afferents were defunctionalized by resiniferatoxin (RTX) pretreatment in BALB/c mice, PGIA was induced by repeated antigen challenges. Hind paw volume, arthritis severity, grasping ability and the mechanonociceptive threshold were monitored during the 17-week experiment. Myeloperoxidase activity, vascular leakage and bone turnover were evaluated by in vivo optical imaging. Bone morphology was assessed using micro-CT, the intertarsal small joints were processed for histopathological analysis. RESULTS: Following desensitization of the capsaicin-sensitive afferents, ankle edema, arthritis severity and mechanical hyperalgesia were markedly diminished. Myeloperoxidase activity was lower in the early, but increased in the late phase, whilst plasma leakage and bone turnover were not altered. Desensitized mice displayed similar bone spurs and erosions, but increased trabecular thickness of the tibia and bony ankylosis of the spine. Intertarsal cartilage thickness was not altered in the model, but desensitization increased this parameter in both the non-arthritic and arthritic groups. CONCLUSION: This is the first integrative in vivo functional and morphological characterization of the PGIA mouse model, wherein peptidergic afferents have an important regulatory function. Their overall effect is proinflammatory by increasing acute inflammation, immune cell activity and pain. Meanwhile, their activation decreases spinal ankylosis, arthritis-induced altered trabecularity, and cartilage thickness in small joints.


Assuntos
Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/patologia , Capsaicina/farmacologia , Proteoglicanas/toxicidade , Fármacos do Sistema Sensorial/farmacologia , Limiar Sensorial/efeitos dos fármacos , Animais , Tornozelo/diagnóstico por imagem , Cartilagem/patologia , Modelos Animais de Doenças , Diterpenos/farmacologia , Feminino , Membro Posterior/efeitos dos fármacos , Membro Posterior/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Neurotoxinas/farmacologia , Peptídeos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Índice de Gravidade de Doença , Coluna Vertebral/diagnóstico por imagem
9.
Int J Mol Sci ; 19(9)2018 Aug 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150589

RESUMO

: Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with diverse developmental roles, including differentiation of skeletal elements. It is a positive regulatory factor of chondrogenesis and osteogenic differentiation in vitro, but little is known about its in vivo role in bone formation. In our experiments, diaphyses of long bones from hind limbs of PACAP gene-deficient mice showed changes in thickness and increased staining intensity. Our main goal was to perform a detailed morphological and molecular biological analysis of femurs from PACAP knockout (KO) and wild type (WT) mice. Transverse diameter and anterior cortical bone thickness of KO femurs showed significant alterations with disturbed Ca2+ accumulation and collagen type I expression. Higher expression and activity of alkaline phosphatase were also observed, accompanied by increased fragility PACAP KO femurs. Increased expression of the elements of bone morphogenic protein (BMP) and hedgehog signalling was also observed, and are possibly responsible for the compensation mechanism accounting for the slight morphological changes. In summary, our results show that lack of PACAP influences molecular and biomechanical properties of bone matrix, activating various signalling cascade changes in a compensatory fashion. The increased fragility of PACAP KO femur further supports the role of endogenous PACAP in in vivo bone formation.


Assuntos
Condrogênese/genética , Osteogênese/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Transdução de Sinais/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Animais , Proteínas Morfogenéticas Ósseas/genética , Proteínas Morfogenéticas Ósseas/metabolismo , Cálcio/metabolismo , Diferenciação Celular/genética , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fêmur/diagnóstico por imagem , Fêmur/metabolismo , Expressão Gênica , Camundongos Knockout , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Microtomografia por Raio-X
10.
Int J Mol Sci ; 19(7)2018 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-29966365

RESUMO

Heterotetrameric N-methyl-d-aspartate type glutamate receptors (NMDAR) are cationic channels primarily permeable for Ca2+. NR1 and NR3 subunits bind glycine, while NR2 subunits bind glutamate for full activation. As NR1 may contain a nuclear localization signal (NLS) that is recognized by importin-α, our aim was to investigate if NMDARs are expressed in the nuclei of melanocytes and melanoma cells. A detailed NMDAR subunit expression pattern was examined by RT-PCRs (reverse transcription followed by polymerase chain reaction), fractionated western blots and immunocytochemistry in human epidermal melanocytes and in human melanoma cell lines A2058, HT199, HT168M1, MEL35/0 and WM35. All kind of NMDAR subunits are expressed as mRNAs in melanocytes, as well as in melanoma cells, while NR2B protein remained undetectable in any cell type. Western blots proved the exclusive presence of NR1 and NR3B in nuclear fractions and immunocytochemistry confirmed NR1-NR3B colocalization inside the nuclei of all melanoma cells. The same phenomenon was not observed in melanocytes. Moreover, protein database analysis revealed a putative NLS in NR3B subunit. Our results support that unusual, NR1-NR3B composed NMDAR complexes are present in the nuclei of melanoma cells. This may indicate a new malignancy-related histopathological feature of melanoma cells and raises the possibility of a glycine-driven, NMDA-related nuclear Ca2+-signalling in these cells.


Assuntos
Melanoma/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Humanos , Melanócitos/metabolismo , Melanoma/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia
11.
J Pathol ; 245(4): 478-490, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29774542

RESUMO

Dysregulation of neuropeptides may play an important role in aging-induced impairments. Among them, pituitary adenylate cyclase-activating polypeptide (PACAP) is a potent cytoprotective peptide that provides an endogenous control against a variety of tissue-damaging stimuli. We hypothesized that the progressive decline of PACAP throughout life and the well-known general cytoprotective effects of PACAP lead to age-related pathophysiological changes in PACAP deficiency, supported by the increased vulnerability to various stressors of animals partially or totally lacking PACAP. Using young and aging CD1 PACAP knockout (KO) and wild type (WT) mice, we demonstrated pre-senile amyloidosis in young PACAP KO animals and showed that senile amyloidosis appeared accelerated, more generalized, more severe, and affected more individuals. Histopathology showed age-related systemic amyloidosis with mainly kidney, spleen, liver, skin, thyroid, intestinal, tracheal, and esophageal involvement. Mass spectrometry-based proteomic analysis, reconfirmed with immunohistochemistry, revealed that apolipoprotein-AIV was the main amyloid protein in the deposits together with several accompanying proteins. Although the local amyloidogenic protein expression was disturbed in KO animals, no difference was found in laboratory lipid parameters, suggesting a complex pathway leading to increased age-related degeneration with amyloid deposits in the absence of PACAP. In spite of no marked inflammatory histological changes or blood test parameters, we detected a disturbed cytokine profile that possibly creates a pro-inflammatory milieu favoring amyloid deposition. In summary, here we describe accelerated systemic senile amyloidosis in PACAP gene-deficient mice, which might indicate an early aging phenomenon in this mouse strain. Thus, PACAP KO mice could serve as a model of accelerated aging with human relevance. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Assuntos
Amiloidose/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Placa Amiloide , Fatores Etários , Amiloidose/genética , Amiloidose/prevenção & controle , Animais , Apolipoproteínas A/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Predisposição Genética para Doença , Mediadores da Inflamação/metabolismo , Camundongos Knockout , Fenótipo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Proteômica/métodos , Índice de Gravidade de Doença , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo
12.
J Vasc Res ; 54(6): 359-366, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29131060

RESUMO

Pituitary adenylate cyclase-activating peptide (PACAP; 1-38 and 1-27) and vasoactive intestinal peptide (VIP) are related neuropeptides of the secretin/glucagon family. Overlapping signaling through G-protein-coupled receptors mediates their vasomotor activity. We previously showed that PACAP deficiency (PACAP-KO) shifts the mechanisms of vascular response and maintains arterial relaxation through the VIP backup mechanism and (mainly) its VPAC1R, but their age-dependent modulation is still unknown. We hypothesized that backup mechanisms exist, which maintain the vasomotor activity of these peptides also in older age. Thus, we investigated the effects of exogenous VIP and PACAP peptides in isolated carotid arteries of 2- and 15-month-old wild-type (WT) and PACAP-KO mice. All peptides induced relaxation in the arteries of young WT mice, whereas in young PACAP-KO mice PACAP1-27 and VIP, but not PACAP1-38, induced relaxation. Unlike VIP, PACAP-induced vasomotor responses were reduced in aging WT mice. However, in the arteries of aging PACAP-KO mice, PACAP1-27- and VIP-induced responses were reduced, but PACAP1-38 showed a greater vasomotor response compared to that of young PACAP-KO animals. There were no significant differences between the vasomotor responses of aging WT and PACAP-KO mice. Our data suggest that, in the absence of PACAP both in young and old ages, the vascular response is mediated through backup mechanisms, most likely VIP, maintaining proper vascular relaxation in aging-induced PACAP insufficiency.


Assuntos
Envelhecimento/metabolismo , Artéria Carótida Primitiva/efeitos dos fármacos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Fatores Etários , Envelhecimento/genética , Animais , Artéria Carótida Primitiva/fisiologia , Relação Dose-Resposta a Droga , Genótipo , Técnicas In Vitro , Masculino , Camundongos Knockout , Fenótipo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo
13.
J Vasc Res ; 54(3): 180-192, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28490016

RESUMO

BACKGROUND: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a multifunctional neuropeptide in the VIP/secretin/glucagon peptide superfamily. Two active forms, PACAP1-38 and PACAP1-27, act through G protein-coupled receptors, the PAC1 and VPAC1/2 receptors. Effects of PACAP include potent vasomotor activity. Vasomotor activity and organ-specific vasomotor effects of PACAP-deficient mice have not yet been investigated; thus, the assessment of its physiological importance in vasomotor functions is still missing. We hypothesized that backup mechanisms exist to maintain PACAP pathway activity in PACAP knockout (KO) mice. Thus, we investigated the vasomotor effects of exogenous vasoactive intestinal peptide (VIP) and PACAP polypeptides in PACAP wild-type (WT) and PACAP-deficient (KO) male mice. METHODS: Carotid and femoral arteries were isolated from 8- to 12-week-old male WT and PACAP-KO mice. Vasomotor responses were measured with isometric myography. RESULTS: In the arteries of WT mice the peptides induced relaxations, which were significantly greater to PACAP1-38 than to PACAP1-27 and VIP. In KO mice, PACAP1-38 did not elicit relaxation, whereas PACAP1-27 and VIP elicited significantly greater relaxation in KO mice than in WT mice. The specific PAC1R and VPAC1R antagonist completely blocked the PACAP-induced relaxations. CONCLUSION: Our data suggest that in PACAP deficiency, backup mechanisms maintain arterial relaxations to polypeptides, indicating an important physiological role for the PACAP pathway in the regulation of vascular tone.


Assuntos
Artéria Carótida Primitiva/efeitos dos fármacos , Artéria Femoral/efeitos dos fármacos , Fragmentos de Peptídeos/farmacologia , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Peptídeo Intestinal Vasoativo/farmacologia , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Artéria Carótida Primitiva/enzimologia , Relação Dose-Resposta a Droga , Artéria Femoral/enzimologia , Genótipo , Técnicas In Vitro , Masculino , Camundongos Knockout , Fenótipo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/agonistas , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/agonistas , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Transdução de Sinais/efeitos dos fármacos
14.
J Mol Neurosci ; 61(4): 468-478, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28168413

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is a multifunctional neuropeptide. In addition to its diverse physiological roles, PACAP has important functions in the embryonic development of various tissues, and it is also considered as a trophic factor during development and in the case of neuronal injuries. Data suggest that the development of the nervous system is severely affected by the lack of endogenous PACAP. Short-term neurofunctional outcome correlates with long-term functional deficits; however, the early neurobehavioral development of PACAP-deficient mice has not yet been evaluated. Therefore, the aim of the present study was to describe the postnatal development of physical signs and neurological reflexes in mice partially or completely lacking PACAP. We examined developmental hallmarks during the first 3 weeks of the postnatal period, during which period most neurological reflexes and motor coordination show most intensive development, and we describe the neurobehavioral development using a complex battery of tests. In the present study, we found that PACAP-deficient mice had slower weight gain throughout the observation period. Interestingly, mice partially lacking PACAP weighed significantly less than homozygous mice. There was no difference between male and female mice during the first 3 weeks. Some other signs were also more severely affected in the heterozygous mice than in the homozygous mice, such as air righting, grasp, and gait initiation reflexes. Interestingly, incisor teeth erupted earlier in mice lacking PACAP. Motor coordination, shown by the number of foot-faults on an elevated grid, was also less developed in PACAP-deficient mice. In summary, our results show that mice lacking endogenous PACAP have slower weight gain during the first weeks of development and slower neurobehavioral development regarding a few developmental hallmarks.


Assuntos
Peso Corporal , Atividade Motora , Sistema Nervoso/crescimento & desenvolvimento , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Reflexo , Animais , Feminino , Heterozigoto , Homozigoto , Incisivo/crescimento & desenvolvimento , Incisivo/metabolismo , Incisivo/ultraestrutura , Masculino , Camundongos , Sistema Nervoso/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/deficiência
15.
Sci Rep ; 7: 39863, 2017 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-28067251

RESUMO

Semicarbazide-sensitive amine oxidase (SSAO) catalyses oxidative deamination of primary amines. Since there is no data about its function in pain and arthritis mechanisms, we investigated the effects of our novel SSAO inhibitor SzV-1287 in chronic mouse models of joint inflammation. Effects of SzV-1287 (20 mg/kg i.p./day) were investigated in the K/BxN serum-transfer and complete Freund's adjuvant (CFA)-evoked active immunization models compared to the reference SSAO inhibitor LJP-1207. Mechanonociception was assessed by aesthesiometry, oedema by plethysmometry, clinical severity by scoring, joint function by grid test, myeloperoxidase activity by luminescence, vascular leakage by fluorescence in vivo imaging, histopathological changes by semiquantitative evaluation, and cytokines by Luminex assay. SzV-1287 significantly inhibited hyperalgesia and oedema in both models. Plasma leakage and keratinocyte chemoattractant production in the tibiotarsal joint, but not myeloperoxidase activity was significantly reduced by SzV-1287 in K/BxN-arthritis. SzV-1287 did not influence vascular and cellular mechanisms in CFA-arthritis, but significantly decreased histopathological alterations. There was no difference in the anti-hyperalgesic and anti-inflammatory actions of SzV-1287 and LJP-1207, but only SzV-1287 decreased CFA-induced tissue damage. Unlike SzV-1287, LJP-1207 induced cartilage destruction, which was confirmed in vitro. SzV-1287 exerts potent analgesic and anti-inflammatory actions in chronic arthritis models of distinct mechanisms, without inducing cartilage damage.


Assuntos
Amina Oxidase (contendo Cobre)/antagonistas & inibidores , Analgésicos não Entorpecentes/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Artrite/tratamento farmacológico , Hidrazinas/uso terapêutico , Articulações/patologia , Oxazóis/uso terapêutico , Oximas/uso terapêutico , Animais , Células Cultivadas , Doença Crônica , Modelos Animais de Doenças , Progressão da Doença , Adjuvante de Freund/imunologia , Humanos , Hidrazinas/farmacologia , Articulações/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Oxazóis/farmacologia , Oximas/farmacologia
16.
Infect Genet Evol ; 45: 402-407, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27717748

RESUMO

Ranaviruses are emerging pathogens associated with high mortality diseases in fish, amphibians and reptiles. Here we describe the whole genome sequence of two ranavirus isolates from brown bullhead (Ameiurus nebulosus) specimens collected in 2012 at two different locations in Hungary during independent mass mortality events. The two Hungarian isolates were highly similar to each other at the genome sequence level (99.9% nucleotide identity) and to a European sheatfish (Silurus glanis) origin ranavirus (ESV, 99.7%-99.9% nucleotide identity). The coding potential of the genomes of both Hungarian isolates, with 136 putative proteins, were shared with that of the ESV. The core genes commonly used in phylogenetic analysis of ranaviruses were not useful to differentiate the two brown bullhead ESV strains. However genome-wide distribution of point mutations and structural variations observed mainly in the non-coding regions of the genome suggested that the ranavirus disease outbreaks in Hungary were caused by different virus strains. At this moment, due to limited whole genome sequence data of ESV it is unclear whether these genomic changes are useful in molecular epidemiological monitoring of ranavirus disease outbreaks. Therefore, complete genome sequencing of further isolates will be needed to identify adequate genetic markers, if any, and demonstrate their utility in disease control and prevention.


Assuntos
Infecções por Vírus de DNA/virologia , Doenças dos Peixes/virologia , Ictaluridae/virologia , Ranavirus/genética , Animais , Infecções por Vírus de DNA/veterinária , Surtos de Doenças , Hungria , Filogenia , Ranavirus/classificação , Ranavirus/isolamento & purificação
17.
Int J Oncol ; 48(3): 983-97, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26717964

RESUMO

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.


Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Ácido Hialurônico/biossíntese , Melanoma/metabolismo , Proteína Fosfatase 2/metabolismo , Calcineurina/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Ciclosporina/química , Flavonoides/química , Glucuronosiltransferase/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Hialuronan Sintases , Sistema de Sinalização das MAP Quinases , Fosfoproteínas/metabolismo , Fosforilação
18.
Int J Mol Sci ; 16(8): 18412-38, 2015 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-26262612

RESUMO

Mature and developing chondrocytes exist in a microenvironment where mechanical load, changes of temperature, osmolarity and acidic pH may influence cellular metabolism. Polymodal Transient Receptor Potential Vanilloid (TRPV) receptors are environmental sensors mediating responses through activation of linked intracellular signalling pathways. In chondrogenic high density cultures established from limb buds of chicken and mouse embryos, we identified TRPV1, TRPV2, TRPV3, TRPV4 and TRPV6 mRNA expression with RT-PCR. In both cultures, a switch in the expression pattern of TRPVs was observed during cartilage formation. The inhibition of TRPVs with the non-selective calcium channel blocker ruthenium red diminished chondrogenesis and caused significant inhibition of proliferation. Incubating cell cultures at 41 °C elevated the expression of TRPV1, and increased cartilage matrix production. When chondrogenic cells were exposed to mechanical load at the time of their differentiation into matrix producing chondrocytes, we detected increased mRNA levels of TRPV3. Our results demonstrate that developing chondrocytes express a full palette of TRPV channels and the switch in the expression pattern suggests differentiation stage-dependent roles of TRPVs during cartilage formation. As TRPV1 and TRPV3 expression was altered by thermal and mechanical stimuli, respectively, these are candidate channels that contribute to the transduction of environmental stimuli in chondrogenic cells.


Assuntos
Condrócitos/metabolismo , Condrogênese , Canais de Cátion TRPV/metabolismo , Animais , Cartilagem/citologia , Cartilagem/fisiologia , Técnicas de Cultura de Células , Células Cultivadas , Embrião de Galinha , Condrócitos/citologia , Condrogênese/efeitos dos fármacos , Temperatura Alta , Camundongos , RNA Mensageiro/genética , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/genética , Transcriptoma , Suporte de Carga
19.
Int J Mol Sci ; 16(8): 17344-67, 2015 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-26230691

RESUMO

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.


Assuntos
Condrócitos/metabolismo , Células-Tronco Embrionárias/metabolismo , Proteínas Hedgehog/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Estresse Mecânico , Animais , Células Cultivadas , Embrião de Galinha , Proteínas Oncogênicas/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/genética , Receptores Tipo II de Peptídeo Intestinal Vasoativo/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/genética , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismo , Transdução de Sinais , Transativadores/metabolismo , Proteína GLI1 em Dedos de Zinco
20.
PLoS One ; 10(4): e0123583, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25893964

RESUMO

The sarcoplasmic/endoplasmic reticulum Ca2+ATPases (SERCAs) are the main Ca2+ pumps which decrease the intracellular Ca2+ level by reaccumulating Ca2+ into the sarcoplasmic reticulum. The neonatal SERCA1b is the major Ca2+ pump in myotubes and young muscle fibers. To understand its role during skeletal muscle differentiation its synthesis has been interfered with specific shRNA sequence. Stably transfected clones showing significantly decreased SERCA1b expression (cloneC1) were selected for experiments. The expression of the regulatory proteins of skeletal muscle differentiation was examined either by Western-blot at the protein level for MyoD, STIM1, calsequestrin (CSQ), and calcineurin (CaN) or by RT-PCR for myostatin and MCIP1.4. Quantitative analysis revealed significant alterations in CSQ, STIM1, and CaN expression in cloneC1 as compared to control cells. To examine the functional consequences of the decreased expression of SERCA1b, repeated Ca2+-transients were evoked by applications of 120 mM KCl. The significantly higher [Ca2+]i measured at the 20th and 40th seconds after the beginning of KCl application (112±3 and 110±3 nM vs. 150±7 and 135±5 nM, in control and in cloneC1 cells, respectively) indicated a decreased Ca2+-uptake capability which was quantified by extracting the maximal pump rate (454±41 µM/s vs. 144±24 µM/s, in control and in cloneC1 cells). Furthermore, the rate of calcium release from the SR (610±60 vs. 377±64 µM/s) and the amount of calcium released (843±75 µM vs. 576±80 µM) were also significantly suppressed. These changes were also accompanied by a reduced activity of CaN in cells with decreased SERCA1b. In parallel, cloneC1 cells showed inhibited cell proliferation and decreased myotube nuclear numbers. Moreover, while cyclosporineA treatment suppressed the proliferation of parental cultures it had no effect on cloneC1 cells. SERCA1b is thus considered to play an essential role in the regulation of [Ca2+]i and its ab ovo gene silencing results in decreased skeletal muscle differentiation.


Assuntos
Cálcio/metabolismo , Diferenciação Celular , Inativação Gênica , Homeostase , Células Musculares/metabolismo , Músculo Esquelético/citologia , Animais , Sinalização do Cálcio , Linhagem Celular , Proliferação de Células , Regulação para Baixo , Camundongos , Modelos Biológicos , Desenvolvimento Muscular , Músculo Esquelético/crescimento & desenvolvimento , RNA Interferente Pequeno/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático , Transfecção
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