RESUMO
The proper regeneration of vessel anastomoses in microvascular surgery is crucial for surgical safety. Pituitary adenylate cyclase-activating polypeptide (PACAP) can aid healing by decreasing inflammation, apoptosis and oxidative stress. In addition to hematological and hemorheological tests, we examined the biomechanical and histological features of vascular anastomoses with or without PACAP addition and/or using a hemostatic sponge (HS). End-to-end anastomoses were established on the right femoral arteries of rats. On the 21st postoperative day, femoral arteries were surgically removed for evaluation of tensile strength and for histological and molecular biological examination. Effects of PACAP were also investigated in tissue culture in vitro to avoid the effects of PACAP degrading enzymes. Surgical trauma and PACAP absorption altered laboratory parameters; most notably, the erythrocyte deformability decreased. Arterial wall thickness showed a reduction in the presence of HS, which was compensated by PACAP in both the tunica media and adventitia in vivo. The administration of PACAP elevated these parameters in vitro. In conclusion, the application of the neuropeptide augmented elastin expression while HS reduced it, but no significant alterations were detected in collagen type I expression. Elasticity and tensile strength increased in the PACAP group, while it decreased in the HS decreased. Their combined use was beneficial for vascular regeneration.
Assuntos
Hemostáticos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Ratos , Animais , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/farmacologia , Hemostáticos/farmacologia , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores Tipo I de Polipeptídeo Intestinal Vasoativo/metabolismoRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP) is a neuropeptide with protective functions in the central nervous system and various peripheral organs. PACAP has the highest expression level in the testes, among the peripheral organs, and has a positive regulative role in spermatogenesis and in sperm motility. In the present study, we explored testicular degenerative alterations in a mouse model of Alzheimer's disease (AD) (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) and demonstrated changes in PACAP-regulated signaling pathways. In addition, the effects of increased physical activity of AD (trained AD (TAD)) mice on testis were also followed. Reduced cell number and decreased thickness of basement membrane were detected in AD samples. These changes were compensated by physical activity. Expression of PACAP receptors and canonical signaling elements such as PKA, P-PKA, PP2A significantly decreased in AD mice, and altered Sox transcription factor expression was also detected. Via this signaling mechanism, physical activity compensated the negative effects of AD on the expression of type IV collagen. Our findings suggest that the testes of AD mice can be a good model of testis degeneration. Moreover, it can be an appropriate organ to follow the effects of various interventions such as physical activity on tissue regeneration and signaling alterations.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Transdução de Sinais/genética , Doenças Testiculares/metabolismo , Testículo/metabolismo , Animais , Contagem de Células , Colágeno Tipo IV/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Condicionamento Físico Animal , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Fatores de Transcrição SOX9/metabolismo , Espermatócitos/metabolismo , Espermatogênese/genética , Espermatogônias/metabolismo , Testículo/patologiaRESUMO
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.
Assuntos
Condrogênese/genética , Receptores de N-Metil-D-Aspartato/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Cálcio/análise , Cálcio/metabolismo , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Galinhas , Condrogênese/efeitos dos fármacos , Ácido Glutâmico/análise , N-Metilaspartato/farmacologia , Receptores de N-Metil-D-Aspartato/agonistas , Transdução de Sinais/efeitos dos fármacosRESUMO
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 TempoRESUMO
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/farmacologiaRESUMO
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 imagemRESUMO
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/fisiologiaRESUMO
: 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-XRESUMO
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 fármacosRESUMO
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/metabolismoRESUMO
Osteoarthritis (OA) is the most common form of chronic musculoskeletal disorders. A migratory stem cell population termed chondrogenic progenitor cells (CPC) with in vitro chondrogenic potential was previously isolated from OA cartilage. Since intracellular Ca(2+) signalling is an important regulator of chondrogenesis, we aimed to provide a detailed understanding of the Ca(2+) homeostasis of CPCs. In this work, CPCs immortalised by lentiviral administration of the human telomerase reverse transcriptase (hTERT) and grown in monolayer cultures were studied. Expressions of all three IP3Rs were confirmed, but no RyR subtypes were detected. Ca(2+) oscillations observed in CPCs were predominantly dependent on Ca(2+) release and store replenishment via store-operated Ca(2+) entry; CPCs express both STIM1 and Orai1 proteins. Expressions of adenosine receptor mRNAs were verified, and adenosine elicited Ca(2+) transients. Various P2 receptor subtypes were identified; P2Y1 can bind ADP; P2Y4 is targeted by UTP; and ATP may evoke Ca(2+) transients via detected P2X subtypes, as well as P2Y1 and P2Y2. Enzymatic breakdown of extracellular nucleotides by apyrase completely abrogated Ca(2+) oscillations, suggesting that an autocrine/paracrine purinergic mechanism may drive Ca(2+) oscillations in these cells. As CPCs possess a broad spectrum of functional molecular elements of Ca(2+) signalling, Ca(2+)-dependent regulatory mechanisms can be supposed to influence their differentiation potential.
Assuntos
Células-Tronco Adultas/metabolismo , Sinalização do Cálcio , Cartilagem/metabolismo , Receptores Purinérgicos/metabolismo , Idoso , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Cartilagem/citologia , Células Cultivadas , Células HEK293 , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1 , Receptores Purinérgicos/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Molécula 1 de Interação EstromalRESUMO
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 CargaRESUMO
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 ZincoRESUMO
Murine micromass models have been extensively applied to study chondrogenesis and osteogenesis to elucidate pathways of endochondral bone formation. Here we provide a detailed comparative analysis of the differentiation potential of micromass cultures established from either BMP-2 overexpressing C3H10T1/2 cells or mouse embryonic limb bud-derived chondroprogenitor cells, using micromass cultures from untransfected C3H10T1/2 cells as controls. Although the BMP-2 overexpressing C3H10T1/2 cells failed to form chondrogenic nodules, cells of both models expressed mRNA transcripts for major cartilage-specific marker genes including Sox9, Acan, Col2a1, Snorc, and Hapln1 at similar temporal sequence, while notable lubricin expression was only detected in primary cultures. Furthermore, mRNA transcripts for markers of osteogenic differentiation including Runx2, Osterix, alkaline phosphatase, osteopontin and osteocalcin were detected in both models, along with matrix calcification. Although the adipogenic lineage-specific marker gene FABP4 was also expressed in micromass cultures, Oil Red O-positive cells along with PPARγ2 transcripts were only detected in C3H10T1/2-derived micromass cultures. Apart from lineage-specific marker genes, pluripotency factors (Nanog and Sox2) were also expressed in these models, reflecting on the presence of various mesenchymal lineages as well as undifferentiated cells. This cellular heterogeneity has to be taken into consideration for the interpretation of data obtained by using these models.
Assuntos
Condrogênese/genética , Botões de Extremidades/embriologia , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética , Adipogenia/genética , Animais , Proteína Morfogenética Óssea 2/biossíntese , Proteína Morfogenética Óssea 2/genética , Diferenciação Celular/fisiologia , Linhagem Celular , Proliferação de Células , Marcadores Genéticos/genética , Camundongos , Camundongos Endogâmicos C3H , RNA Mensageiro/biossíntese , Transdução de Sinais/genéticaRESUMO
Articular cartilage is characterized by a low density of chondrocytes surrounded by an abundant extracellular matrix (ECM) consisting of a dense mixture of collagens, proteoglycans, and glycosaminoglycans. Due to its low cellularity and high proteoglycan content, it is particularly challenging to extract high-quality total RNA suitable for sensitive high-throughput downstream applications such as RNA sequencing (RNA-Seq). Available protocols for high-quality RNA isolation from articular chondrocytes are inconsistent, resulting in suboptimal yield and compromised quality. This poses a significant difficulty in the application of RNA-Seq to study the cartilage transcriptome. Current protocols rely either on dissociation of cartilage ECM by collagenase digestion or pulverizing cartilage using various methods prior to RNA extraction. However, protocols for cartilage processing vary significantly depending on the species and source of cartilage within the body. Protocols for isolating RNA from human or large mammal (e.g., horse or cattle) cartilage samples are available, but this is not the case for chicken cartilage, despite the species being extensively used in cartilage research. Here, we present two improved RNA isolation protocols based on pulverization of fresh articular cartilage using a cryogenic mill or on enzymatic digestion using 1.2% (w/v) collagenase II. Our protocols optimize the collection and tissue processing steps to minimize RNA degradation and enhance RNA purity. Our results show that RNA purified from chicken articular cartilage using these methods has appropriate quality for RNA-Seq experiments. The procedure is applicable for RNA extraction from cartilage from other species such as dog, cat, sheep, and goat. The workflow for RNA-Seq analysis is also described here. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Extraction of total RNA from pulverized chicken articular cartilage Alternate Protocol: Extraction of total RNA from collagen-digested articular cartilage Support Protocol: Dissection of chicken articular cartilage from the knee joint Basic Protocol 2: RNA sequencing of total RNA from chicken articular cartilage.
Assuntos
Cartilagem Articular , Humanos , Animais , Bovinos , Cães , Cavalos , Ovinos , Sequenciamento de Nucleotídeos em Larga Escala , Condrócitos , Galinhas/genética , Cabras , Proteoglicanas , RNA/genéticaRESUMO
Much of the skeletal system develops by endochondral ossification, a process that takes place in early fetal life. This makes the early stages of chondrogenesis, i.e., when chondroprogenitor mesenchymal cells differentiate to chondroblasts, challenging to study in vivo. In vitro methods for the study of chondrogenic differentiation have been available for some time. There is currently high interest in developing fine-tuned methodology that would allow chondrogenic cells to rebuild articular cartilage and restore joint functionality. The micromass culture system that relies on embryonic limb bud-derived chondroprogenitor cells is a popular method for the study of the signaling pathways that control the formation and maturation of cartilage. In this protocol, we describe a technique fine-tuned in our laboratory for culturing limb bud-derived mesenchymal cells from early-stage chick embryos in high density (Basic Protocol 1). We also provide a fine-tuned method for high-efficiency transient transfection of cells before plating using electroporation (Basic Protocol 2). In addition, protocols for histochemical detection of cartilage extracellular matrix using dimethyl methylene blue, Alcian blue, and safranin O are also provided (Basic Protocol 3 and Alternate Protocols 1 and 2, respectively). Finally, a step-by-step guide on a cell viability/proliferation assay using MTT reagent is also described (Basic Protocol 4). © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Micromass culture of chick embryonic limb bud-derived cells Basic Protocol 2: Transfection of cells with siRNA constructs using electroporation prior to micromass culturing Basic Protocol 3: Qualitative and quantitative assessment of cartilage matrix production using dimethyl methylene blue staining and image analysis Alternate Protocol 1: Qualitative assessment of cartilage matrix production using Alcian blue staining Alternate Protocol 2: Qualitative assessment of cartilage matrix production using safranin O staining Basic Protocol 4: Measurement of mitochondrial activity with the MTT assay.
Assuntos
Galinhas , Azul de Metileno , Animais , Embrião de Galinha , Azul de Metileno/metabolismo , Azul Alciano/metabolismo , Células Cultivadas , Cartilagem/metabolismo , RegeneraçãoRESUMO
Introduction: Valve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Methods and result: Using in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics [desferrioxamine, CoCl2, Daprodustat (DPD)] promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted aortic VC in the CKD mice model. Discussion: HIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate aortic VC.
RESUMO
Endometriosis is a chronic gynecological disease that causes numerous severe symptoms in affected women. Revealing alterations of the molecular processes in ectopic endometrial tissue is the current policy for understanding the pathomechanisms and discovering potential novel therapeutic targets. Examining molecular processes of eutopic endometrium is likely to be a convenient method to compare it with the molecular alterations observed in ectopic tissues. The aim of the present study was to determine what proportion of the surgically resected eutopic endometrial samples is suitable for further experiments so that these can be comparable with endometriosis. Final hospital reports and histopathology reports of a 3-year-long period (1162 cases) were analysed. The application of a retrospective screening method promoted the categorization of these cases, and quantification of the categorized cases was accomplished. In addition, results obtained from cultured endometrium samples were also detailed. Only a small number of the harvested endometrial samples was suitable for further molecular analysis, while preoperative screening protocol could enlarge this fraction. Applying clinical and histopathological selection and exclusion criteria for tissue screening and histopathological examination of samples could ensure the comparability of healthy endometrium with endometriosis. The present study could be useful for researchers who intend to perform molecular experiments to compare endometriosis with the physiological processes of the endometrium.
RESUMO
BACKGROUND: Alzheimer's disease (AD) is a neurodegenerative illness, with several peripheral pathological signs such as accumulation of amyloid-ß (Aß) plaques in the kidney. Alterations of transforming growth factor ß (TGFß) signaling in the kidney can induce fibrosis, thus disturbing the elimination of Aß. OBJECTIVE: A protective role of increased physical activity has been proven in AD and in kidney fibrosis, but it is not clear whether TGFß signalization is involved in this effect. METHODS: The effects of long-term training on fibrosis were investigated in the kidneys of mice representing a model of AD (B6C3-Tg(APPswe,PSEN1dE9)85Dbo/J) by comparing wild type and AD organs. Alterations of canonical and non-canonical TGFß signaling pathways were followed with PCR, western blot, and immunohistochemistry. RESULTS: Accumulation of collagen type I and interstitial fibrosis were reduced in kidneys of AD mice after long-term training. AD induced the activation of canonical and non-canonical TGFß pathways in non-trained mice, while expression levels of signal molecules of both TGFß pathways became normalized in trained AD mice. Decreased amounts of phosphoproteins with molecular weight corresponding to that of tau and the cleaved C-terminal of AßPP were detected upon exercising, along with a significant increase of PP2A catalytic subunit expression. CONCLUSION: Our data suggest that physical training has beneficial effects on fibrosis formation in kidneys of AD mice and TGFß signaling plays a role in this phenomenon.
Assuntos
Doença de Alzheimer/patologia , Rim/patologia , Condicionamento Físico Animal/fisiologia , Transdução de Sinais/fisiologia , Fator de Crescimento Transformador beta/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Fibrose/metabolismo , Fibrose/patologia , Rim/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Placa Amiloide/metabolismo , Placa Amiloide/patologiaRESUMO
Pituitary adenylate cyclase activating polypeptide (PACAP) is an endogenous neuropeptide which is distributed throughout the body. PACAP influences development of various tissues and exerts protective function during cellular stress and in some tumour formation. No evidence is available on its role in neural crest derived melanocytes and its malignant transformation into melanoma. Expression of PACAP receptors was examined in human skin samples, melanoma lesions and in a primary melanocyte cell culture. A2058 and WM35 melanoma cell lines, representing two different stages of melanoma progression, were used to investigate the effects of PACAP. PAC1 receptor was identified in melanocytes in vivo and in vitro and in melanoma cell lines as well as in melanoma lesions. PACAP administration did not alter viability but decreased proliferation of melanoma cells. With live imaging random motility, average speed, vectorial distance and maximum distance of migration of cells were reduced upon PACAP treatment. PACAP administration did not alter viability but decreased proliferation capacity of melanoma cells. On the other hand, PACAP administration decreased the migration of melanoma cell lines towards fibronectin chemoattractant in the Boyden chamber. Furthermore, the presence of the neuropeptide inhibited the invasion capability of melanoma cell lines in Matrigel chambers. In summary, we provide evidence that PACAP receptors are expressed in melanocytes and in melanoma cells. Our results also prove that various aspects of the cellular motility were inhibited by this neuropeptide. On the basis of these results, we propose PACAP signalling as a possible target in melanoma progression.