RESUMO
Isobaric heat capacities C p of benzene confined in silica MCM-41 mesopores with average diameters equal to and smaller than 2.9 nm were measured by precise adiabatic calorimetry. The confined benzene samples revealed no thermal anomaly due to crystallization/fusion and vitrified at low temperatures. The C p curves displayed a hump and a considerably quick decrease on the low-temperature side of the hump as the pore diameter increased. The enthalpy-relaxation effects observed on intermittent heating showed that the anomaly of the C p hump and quick decrease is not assigned to a glass transition. The bend in the temperature dependence of density reported previously was interpreted as corresponding to the quick decrease in C p . We concluded that the anomalous C p and density behaviors originated from the ordering/excitation in the configurational state, close to the ground state, of confined molecular aggregate and proposed a scenario that explains the general C p curves of ordinary bulk supercooled liquids in equilibrium at low temperatures below the glass-transition temperatures.
RESUMO
Poly(ADP-ribose) glycohydrolase (PARG) is the primary enzyme responsible for the degradation of poly(ADP-ribose). PARG dysfunction sensitizes cells to alkylating agents and induces cell death; however, the details of this effect have not been fully elucidated. Here, we investigated the mechanism by which PARG deficiency leads to cell death in different cell types using methylmethanesulfonate (MMS), an alkylating agent, and Parg(-/-) mouse ES cells and human cancer cell lines. Parg(-/-) mouse ES cells showed increased levels of γ-H2AX, a marker of DNA double strand breaks (DSBs), accumulation of poly(ADP-ribose), p53 network activation, and S-phase arrest. Early apoptosis was enhanced in Parg(-/-) mouse ES cells. Parg(-/-) ES cells predominantly underwent caspase-dependent apoptosis. PARG was then knocked down in a p53-defective cell line, MIAPaCa2 cells, a human pancreatic cancer cell line. MIAPaCa2 cells were sensitized to MMS by PARG knockdown. Enhanced necrotic cell death was induced in MIAPaCa2 cells after augmenting γ-H2AX levels and S-phase arrest. Taken together, these data suggest that DSB repair defect causing S-phase arrest, but p53 status was not important for sensitization to alkylation DNA damage by PARG dysfunction, whereas the cell death pathway is dependent on the cell type. This study demonstrates that functional inhibition of PARG may be useful for sensitizing at least particular cancer cells to alkylating agents.
Assuntos
Apoptose , Adutos de DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Glicosídeo Hidrolases/genética , Fase S , Alquilação , Animais , Antineoplásicos Alquilantes/farmacologia , Caspases/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Técnicas de Inativação de Genes , Glicosídeo Hidrolases/deficiência , Humanos , Potencial da Membrana Mitocondrial , Metanossulfonato de Metila/farmacologia , Camundongos , Mutagênicos/farmacologia , Poli Adenosina Difosfato Ribose/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
We evaluated our results of video-assisted thoracic surgery (VATS) performed for lung cancer over 8 years. Between April 2000 and October 2008, a total of 409 (60.9%) underwent VATS for lung cancer. Operative procedures as a radical operation were partial resection in 58 patients, segmentectomy in 64 patients, and lobectomy in 229 patients. There was 1 patient with operative death including hospital death due to pulmonary thromboembolism. In a median follow-up period of 21 months, the 5-year cause specific survival rate was 93.7%. According to operative procedures, the 5-year survival rate was 100% in patients underwent partial resection and segmentectomy, and 91.1% in patients underwent lobectomy. According to pathological stages, the 5-year survival rate was 98.8% in 289 patients with stage IA, 69.1% in 34 patients with stage IB, and 68.2% in 14 patients with stage IIIA. In conclusion, VATS lobectomy and VATS intentional limited resection can be performed with low mortality and good prognosis for clinical stage IA lung cancer patients.
Assuntos
Adenocarcinoma/cirurgia , Carcinoma de Células Escamosas/cirurgia , Neoplasias Pulmonares/cirurgia , Pneumonectomia , Carcinoma de Pequenas Células do Pulmão/cirurgia , Cirurgia Torácica Vídeoassistida , Adenocarcinoma/patologia , Carcinoma de Células Escamosas/patologia , Humanos , Neoplasias Pulmonares/patologia , Estadiamento de Neoplasias , Pneumonectomia/métodos , Pneumonectomia/mortalidade , Prognóstico , Carcinoma de Pequenas Células do Pulmão/patologia , Taxa de Sobrevida , Cirurgia Torácica Vídeoassistida/métodos , Cirurgia Torácica Vídeoassistida/mortalidadeRESUMO
The aim of this study was to evaluate serum midkine (S-MK) concentrations as a prognostic tumour marker in oral squamous cell carcinoma (OSCC). We measured S-MK concentrations in patients with OSCC and healthy volunteers. In addition, we performed real-time quantitative reverse transcription-PCR analysis and immunohistochemistry with fresh tumour samples. To determine whether S-MK concentrations have prognostic value, we performed survival analyses with clinical information by using the log-rank test. Serum midkine concentrations were significantly higher in patients with OSCC than in healthy controls (P<0.001). Serum midkine concentrations were also significantly increased in early-stage OSCC compared with those of healthy individuals (P<0.001). In addition, immunohistochemistry allowed identification of overexpressed MK protein in OSCC tissues. MK mRNA showed higher expression in OSCC samples compared with normal mucosal samples. Patients in high S-MK groups showed a significantly lower 5-year survival rate compared with patients in low S-MK groups (P<0.05). The increased S-MK concentrations in early-stage OSCC were strongly associated with poor survival. Serum midkine concentrations may thus be a useful marker not only for cancer screening but also for predicting prognosis of OSCC patients.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma de Células Escamosas/sangue , Citocinas/sangue , Neoplasias Bucais/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Midkina , Mucosa Bucal/metabolismo , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Estadiamento de Neoplasias , Prognóstico , RNA Mensageiro/sangue , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Taxa de SobrevidaRESUMO
Migration of intraepithelial lymphocytes (IELs) into intestinal epithelium is not yet well understood. We established an IEL-cell line from ovalbumin (OVA) 23-3 transgenic (Tg) mice and investigated the effect of antigen stimulation on the dynamic process of IEL migration into small intestinal mucosa. The cell line was a T cell receptor (TCR) alphabeta(+) CD4(+) CD8(-) phenotype, expressing alphaEbeta7 integrin in 90% of cells. Under intravital microscopy, the lined IELs adhered selectively to the microvessels of the intestinal villus tip of the Tg mice. The accumulation of IELs was significantly inhibited by an antibody against beta7-integrin and MAdCAM-1. When IELs were stimulated with OVA, the accumulation was attenuated compared to that of resting cells, with decreased expression of alphaEbeta7 integrin. In Tg mice fed with OVA, the number of IELs which migrated in the villus mucosa was significantly smaller than in the non-fed controls. The preferential migratory capacity of IELs to villus mucosa may be altered by specific antigen stimulations.
Assuntos
Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Subpopulações de Linfócitos T/imunologia , Animais , Antígenos/imunologia , Antígenos de Superfície/metabolismo , Adesão Celular/imunologia , Moléculas de Adesão Celular/imunologia , Linhagem Celular , Movimento Celular/imunologia , Imunidade nas Mucosas , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/imunologiaRESUMO
The objective of this study was to determine whether specific adhesion molecules modulate lymphocyte movement from Peyer's patches into intestinal microlymphatics. The fluorochrome acridine orange was injected via a micropipette into Peyer's patches to fill lymphatics. The flux of labeled lymphocytes into intestinal microlymphatics was monitored with intravital fluorescence microscopy. The lymphatic microvessels in the perifollicular area of Peyer's patches were filled with lymphocytes, most of which remained within the lymphatics. Some lymphocytes became detached and were drained into intestinal lymph. Administration of antibodies directed against ICAM-1 significantly increased lymphocyte flux into interfollicular lymphatics. The immunohistochemical study showed intense ICAM-1 expression on the lymphocytes densely packed in the lymphatics surrounding follicles in Peyer's patches. A large number of lymphocytes are normally sequestered in the lymphatic network of Peyer's patches. This sequestration of lymphocytes is largely mediated by ICAM-1-dependent cell-cell interactions.
Assuntos
Movimento Celular/imunologia , Molécula 1 de Adesão Intercelular/imunologia , Linfócitos/imunologia , Nódulos Linfáticos Agregados/imunologia , Animais , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , Linfócitos/patologia , Masculino , Mesentério/imunologia , Mesentério/patologia , Nódulos Linfáticos Agregados/patologia , Ratos , Ratos WistarRESUMO
The recirculation of lymphocytes through the intestinal mucosa is important for specific immune defense, but the origin and differentiation of intraepithelial lymphocytes (IEL) are not fully understood. The present study therefore used intravital microscopy to investigate the migration of IEL to the villus mucosa and Peyer's patches of the small intestine. IEL were separated from inverted murine small intestine and mesenteric lymph node (MLN) T cells were also isolated. The adhesion of fluorescence-labeled lymphocytes to postcapillary venules (PCV) of Peyer's patches and arcade microvessels of small intestinal villi was observed after injection. In some experiments, the effect of antibodies against adhesion molecules on cell kinetics were investigated. IEL time-dependently accumulated in villus microvessels of the small intestine, whereas few MLN cells did. Few IEL adhered to the PCV of Peyer's patches. IEL were shown to express alpha(E)beta(7)-integrin but not L-selectin. The accumulation of IEL in villus archade was significantly inhibited by antibody against beta(7)-integrin or mucosal addressin cell adhesion molecules (MAdCAM)-1, but not by alpha(E)-integrin. The combined blocking of beta(7)-integrin and MAdCAM-1 further attenuated the sticking of IEL in this area, although it did not entirely block the IEL adherence. The adherence of CD4(+) or TCRalphabeta IEL to villus microvessels was significantly greater than that of CD4(-) or TCRgammadelta IEL. It was demonstrated in situ for the first time that IEL adhered selectively to the villus microvessels of the small intestine partly via beta(7) and MAdCAM-1.
Assuntos
Cadeias alfa de Integrinas , Cadeias beta de Integrinas , Mucosa Intestinal/imunologia , Intestino Delgado/imunologia , Linfócitos/imunologia , Microcirculação/imunologia , Animais , Antígenos CD , Antígenos CD4 , Adesão Celular , Moléculas de Adesão Celular , Diferenciação Celular , Movimento Celular , Feminino , Imunoglobulinas , Integrinas , Mucosa Intestinal/citologia , Intestino Delgado/irrigação sanguínea , Intestino Delgado/citologia , Linfonodos/citologia , Linfonodos/imunologia , Subpopulações de Linfócitos/citologia , Subpopulações de Linfócitos/imunologia , Linfócitos/citologia , Masculino , Mesentério/citologia , Mesentério/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microcirculação/citologia , Mucoproteínas , Nódulos Linfáticos Agregados/irrigação sanguínea , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta , Receptores de Retorno de LinfócitosRESUMO
BACKGROUND/AIMS: Cytokine-induced neutrophil chemoattractant (CINC/gro), a member of interleukin-8 family, was found as a potent chemotactic factor for rat neutrophils. Although several chemokines have been shown to be potent regulators of T cell chemotaxis in vitro, the potential role of chemokines in T-cell migration in gut-associated lymphoid tissues has not been investigated in vivo. In the present study, the effects of CINC/gro on T-lymphocyte migration were examined in rat Peyer's patches. METHODS: T lymphocytes collected from intestinal lymph of rats were fluorescence-labeled and injected into the jugular vein. Peyer's patches of the recipient rats were observed with intravital fluorescence microscopy and the effects of CINC/gro infusion was investigated. Lymphocyte flux in mesenteric collecting lymphatics was also observed. RESULTS: In vivo infusion of CINC/gro significantly attenuated the initial lymphocyte interaction with postcapillary venules of Peyer's patches. However, once these lymphocytes adhered to venules, CINC/gro treatment significantly accelerated the transendothelial migration of T lymphocytes and they also significantly increased their subsequent flux in collecting lymphatics. CONCLUSION: There is a possibility that CINC/gro could modulate the characteristics of T lymphocyte homing in the inflammatory sites of gut.
Assuntos
Movimento Celular , Quimiocinas CXC , Fatores Quimiotáticos/farmacologia , Quimiotaxia , Inibidores do Crescimento/farmacologia , Substâncias de Crescimento/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Intestino Delgado/imunologia , Nódulos Linfáticos Agregados/imunologia , Linfócitos T/fisiologia , Animais , Adesão Celular , Quimiocina CXCL1 , Inflamação , Intestino Delgado/patologia , Sistema Linfático , Masculino , Ratos , Ratos WistarRESUMO
The binding of advanced glycation end products (AGE) to the receptor for AGE (RAGE) is known to deteriorate various cell functions and is implicated in the pathogenesis of diabetic vascular complications. Here we show that AGE, tumor necrosis factor-alpha (TNF-alpha), and 17beta-estradiol (E(2)) up-regulated RAGE mRNA and protein levels in human microvascular endothelial cells and ECV304 cells, with the mRNA stability being essentially invariant. Transient transfection experiments with human RAGE promoter-luciferase chimeras revealed that the region from nucleotide number -751 to -629 and the region from -239 to -89 in the RAGE 5'-flanking sequence exhibited the AGE/TNF-alpha and E(2) responsiveness, respectively. Site-directed mutation of an nuclear factor-kappaB (NF-kappaB) site at -671 or of Sp-1 sites at -189 and -172 residing in those regions resulted in an abrogation of the AGE/TNF-alpha- or E(2)-mediated transcriptional activation. Electrophoretic mobility shift assays revealed that ECV304 cell nuclear extracts contained factors which retarded the NF-kappaB and Sp-1 elements, and that the DNA-protein complexes were supershifted by anti-p65/p50 NF-kappaB and anti-Sp-1/estrogen receptor alpha antibodies, respectively. These results suggest that AGE, TNF-alpha, and E(2) can activate the RAGE gene through NF-kappaB and Sp-1, causing enhanced AGE-RAGE interactions, which would lead to an exacerbation of diabetic microvasculopathy.
Assuntos
Estradiol/metabolismo , Produtos Finais de Glicação Avançada/metabolismo , NF-kappa B/metabolismo , Receptores Imunológicos/metabolismo , Fator de Transcrição Sp1/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Escherichia coli/metabolismo , Humanos , Microcirculação/metabolismo , Mutagênese Sítio-Dirigida , Regiões Promotoras Genéticas , Ligação Proteica , RNA Mensageiro/metabolismo , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Tempo , Transcrição Gênica , Ativação Transcricional , Transfecção , Regulação para CimaRESUMO
Although activation of lymphocytes is known to be associated with profound changes in homing behavior, it remains unclear how activation alters migration of gut-derived lymphocytes in lymphoid and nonlymphoid organs. The objectives of this study were 1) to compare migration of naive and concanavalin A (ConA)-activated T lymphocytes into the gut mucosa, spleen, and liver and 2) to define the role of specific adhesion molecules in this homing process. Fluorescently labeled T lymphocytes collected from rat intestinal lymph were injected into the jugular vein, and the kinetics of appearance of the infused lymphocytes were monitored in ileal Peyer's patches, spleen, and liver. The migration of naive and ConA-activated T lymphocytes into microvessels were compared using an intravital microscope. ConA stimulation significantly increased the rolling velocity of T lymphocytes in postcapillary venules of Peyer's patches, and ConA-stimulated lymphocytes exhibited a loss of the selective adherence properties in Peyer's patches that is normally observed with naive T cells. ConA activation also suppressed the accumulation of T cells in the spleen. On the other hand, the adherence of T cells to hepatic sinusoidal endothelium was significantly increased after ConA activation, especially in the periportal area, and this increase was attenuated by an anti-intercellular adhesion molecule (ICAM)-1 antibody. Flow cytometry analysis revealed a decline in L-selectin expression and an increase in CD11a expression and ICAM-1 on the surface of ConA-treated T cells. In conclusion, activation of gut-derived T lymphocytes with ConA significantly alters their migration path, with a diminished localization to Peyer's patches and spleen and a preferential accumulation in hepatic sinusoids. This altered migration pattern likely results from changes in the expression of leukocyte adhesion molecules such as L-selectin and CD11a.
Assuntos
Concanavalina A/farmacologia , Intestinos/citologia , Ativação Linfocitária/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/fisiologia , Animais , Anticorpos/farmacologia , Moléculas de Adesão Celular/imunologia , Moléculas de Adesão Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Fígado/citologia , Masculino , Ratos , Ratos Wistar , Baço/citologia , Linfócitos T/metabolismoRESUMO
Brain water diffusion in response to transient global ischemia (12 min), reperfusion (60 min), and cardiac arrest was monitored by localized proton magnetic resonance spectroscopy. The trace of the apparent diffusion coefficient tensor (ADC(Av)) was determined at high temporal resolution (10 sec) to assess the putative neuroprotective potential of oral creatine (Cr) in rats that received 2.2 g Cr-monohydrate per kg body weight per day for 10 days (n = 8) relative to controls (n = 9). Cr-fed rats revealed a statistically significant increase of the cerebral concentration ratio of Cr to choline-containing compounds (20%). The decrease of the ADC(Av) value during acute ischemia showed a three-phasic behavior in line with energy depletion, cytotoxic edema, and brain cooling. In Cr-fed rats, slightly less severe and mildly delayed diffusion changes during ischemia and similar beneficial trends during early reperfusion did not reach statistical significance. Magn Reson Med 42:798-802, 1999.
Assuntos
Água Corporal/metabolismo , Encéfalo/metabolismo , Creatina/farmacologia , Ataque Isquêmico Transitório/metabolismo , Animais , Química Encefálica , Difusão , Metabolismo Energético , Espectroscopia de Ressonância Magnética , Masculino , Ratos , Ratos WistarRESUMO
Proton magnetic resonance spectroscopy (MRS) was employed to determine the concentrations of N-acetylaspartate (NAA), total creatine (tCr), choline-containing compounds (Cho), myo-inositol (Ins), glucose (Glc), and lactate (Lac) in rat brain before and after 10 days of oral supplementation of 2.6 g Cr-monohydrate per kg body weight per day. Measurements were performed both in vitro (n = 16) and in vivo (n = 6). The neuroprotective potential of oral Cr was assessed by dynamically monitoring brain Glc and Lac in response to transient global ischemia (12 min). In comparison to controls the in vitro concentrations of Cr (13.1 +/- 9.3%) and Ins (12.7 +/- 14. 0%) were significantly increased in Cr-fed rats. Under in vivo conditions, the data revealed trends for elevated tCr (4.7%) and Ins (10.6%) which were enhanced in the concentration ratios of tCr:Cho (10.2%) and Ins:Cho (17.8%). Together with an increased Glc level (27.3%), the observation of a statistically significant decrease of brain Lac (-38.5 +/- 19.3%) in Cr-fed rats may reflect a shift of the energy metabolism from non-oxidative toward oxidative glycolysis. One hour after global ischemia most of the metabolic differences between Cr-fed rats and controls were retained. The increased Glc level (44.4 +/- 33.3%) reached statistical significance, but the accumulation of Lac and its time course during ischemia and early reperfusion showed no differences between Cr-fed rats and controls.
Assuntos
Encéfalo/metabolismo , Creatina/farmacologia , Suplementos Nutricionais , Ataque Isquêmico Transitório/dietoterapia , Ataque Isquêmico Transitório/metabolismo , Fármacos Neuroprotetores/farmacologia , Administração Oral , Anaerobiose , Animais , Encéfalo/irrigação sanguínea , Creatina/farmacocinética , Modelos Animais de Doenças , Glicólise/efeitos dos fármacos , Espectroscopia de Ressonância Magnética/métodos , Masculino , Prótons , Ratos , Ratos WistarRESUMO
Protocols suitable for repeated magnetic resonance imaging (MRI) studies of the tree shrew's brain were established. This included the development of (i) a technique for prolonged inhalation anesthesia by endotracheal intubation; (ii) a reproducible fixation of the animal's head in a stereotaxic frame and finally (iii) the set-up of the hardware (rf coil) and software (MRI sequences) of the MRI system. The endotracheal intubation as well as the repeated and prolonged anesthesia showed no complications. The in vivo measurements of the tree shrew's hippocampal formation revealed a high reproducibility. Right and left hippocampal volume was determined as 85.2 mm3 +/- 8% and 87.4 mm3 +/- 10%, respectively. The utility of MRI in delineating alterations in brain anatomy was demonstrated in three animals receiving cortisol via the drinking water (5 mg/animal/day). After a 4-week treatment, in two of the three tree shrews a reduction in hippocampal volume was observed. Thus, the MRI protocols used here allow for repeated and non-invasive measurements of changes in hippocampal anatomy within the same animal and to monitor the temporal dynamics of structural alterations within this brain structure.
Assuntos
Hipocampo/anatomia & histologia , Imageamento por Ressonância Magnética/métodos , Animais , Anti-Inflamatórios/farmacologia , Hipocampo/efeitos dos fármacos , Hidrocortisona/farmacologia , Imageamento por Ressonância Magnética/instrumentação , Masculino , TupaiidaeRESUMO
We experienced a case of solitary trigeminal neurinoma in a 10-year-old boy without stigmata of neurofibromatosis. This boy presented with progressive cerebellar ataxia, right abducens palsy and right hemiparesis. Magnetic resonance images (MRI) demonstrated a tumour extending into both the middle and posterior fossas forming a dumbbell-shaped mass lesion with a size of 35 x 30 x 45 mm. The tumour was successfully removed free of complications by a two-stage operation composed of right subtemporal and suboccipital retromastoid approaches. It is extremely rare for a solitary neurinoma to occur at this site in a child without neurofibromatosis. Surgical approaches in this type of tumour are discussed with a review of the literature.
Assuntos
Neoplasias dos Nervos Cranianos/cirurgia , Neurilemoma/cirurgia , Nervo Trigêmeo/cirurgia , Criança , Neoplasias dos Nervos Cranianos/patologia , Humanos , Imageamento por Ressonância Magnética , Masculino , Neurilemoma/patologia , Resultado do TratamentoRESUMO
Insulin treatment is known epidemiologically as an independent risk factor for the progression of diabetic retinopathy. However, how insulin exacerbates the retinopathy is not yet fully understood. In this study, we investigate the effects of insulin on the growth and tube formation of microvascular endothelial cells (EC). When human skin microvascular EC were grown under various concentrations of insulin, DNA synthesis as well as tube formation of EC was found to be significantly stimulated. We obtained evidence that it is mainly vascular endothelial growth factor (VEGF) that mediates the angiogenic activity of insulin as follows. (1) Insulin upregulates the level of mRNA coding for secretory forms of VEGF, while the expression of the two VEGF receptor genes, kinase insert domain-containing receptor (kdr) and fms-like tyrosine kinase1 (flt1), was essentially unchanged by exposure to insulin. (2) A monoclonal antibody against human VEGF can completely neutralize both the proliferation and the tube formation of EC induced by insulin. The angiogenic effects of insulin were additive with those of hypoxia, a principal factor that causes angiogenesis. Further, insulin significantly stimulated plasminogen activator inhibitor-1 activity in EC. The results thus suggest that insulin not only elicits angiogenesis through the induction of autocrine VEGF but also is a predisposing factor for thrombogenesis, which may give rise to focal ischemia that could superdrive angiogenesis, thereby leading to the exacerbation of diabetic retinopathy.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Hipoglicemiantes/farmacologia , Insulina/farmacologia , Linfocinas/fisiologia , Microcirculação , Comunicação Autócrina/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Desenvolvimento Embrionário e Fetal , Humanos , Proteínas Recombinantes/farmacologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Vascular calcification in advanced atherosclerosis is frequently associated with diabetes, and is a predictor of future cardiovascular events. To investigate the molecular mechanisms of vascular calcification, we examined whether advanced glycation endproducts (AGE) formed at an accelerated rate under diabetes induce the osteoblastic differentiation of pericytes, a mesenchymal progenitor. First, von Kossa staining demonstrated that AGE significantly increased the number of calcified nodules in a bovine pericyte culture. AGE were also found to induce calcium accumulation in the pericyte monolayer in time- and dose-dependent manners. Second, quantitative reverse transcription-polymerase chain reaction revealed that AGE increased the pericyte levels of mRNAs coding for alkaline phosphatase and osteopontin, the representative markers for early and late osteoblastic differentiation, respectively. Alkaline phosphatase activity was actually enhanced by AGE. The results suggest that AGE have the ability to induce the osteoblatic differentiation of pericytes, which would contribute to the development of vascular calcification in diabetes.
Assuntos
Calcinose , Produtos Finais de Glicação Avançada , Vasos Retinianos/patologia , Fosfatase Alcalina/genética , Animais , Sequência de Bases , Cálcio/metabolismo , Bovinos , Primers do DNA , Osteopontina , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Vasos Retinianos/enzimologia , Vasos Retinianos/metabolismo , Sialoglicoproteínas/genéticaAssuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Cafeína/administração & dosagem , Neoplasias do Colo/tratamento farmacológico , Leiomiossarcoma/tratamento farmacológico , Cisplatino/administração & dosagem , Neoplasias do Colo/diagnóstico por imagem , Doxorrubicina/administração & dosagem , Feminino , Humanos , Leiomiossarcoma/diagnóstico por imagem , Pessoa de Meia-Idade , Tomografia Computadorizada por Raios XRESUMO
Angiogenesis is the process by which new vascular networks are formed from preexisting capillaries. The small vessels are composed of two types of cells, namely endothelial cells (EC) and pericytes, with the former being encircled by the latter. We previously showed that hypoxia, the principal cause of angiogenesis, can induce the proliferation of pericytes as well as EC. In this report we present evidence that the hypoxic induction of pericyte growth can be ascribed at least in part to vascular endothelial growth factor (VEGF) produced by this very cell type. First, the finding that hypoxia can stimulate the proliferation of pericytes was confirmed by cultivating bovine retinal pericytes in a controlled-atmosphere culture chamber containing various concentrations of oxygen and then assaying pericyte synthesis of DNA. Second, Northern blot analysis revealed that pericyte levels of mRNA encoding VEGF increased as the atmospheric oxygen tension was decreased; this was accompanied by an increase in de novo synthesis of VEGF proteins. Third, pericytes were able to respond to exogenously added VEGF, resulting in a dose-dependent increase in viable cell numbers. Fourth, polyclonal antibodies against VEGF efficiently blocked the hypoxic induction of pericyte growth. Fifth, pericytes expressed the gene for fms-like tyrosine kinase 1 (flt1) as the predominant form of VEGF receptor, and tyrosine phosphorylation of this receptor protein was enhanced when pericytes were exposed to hypoxia, as it was when cells were exposed to VEGF. Sixth, the antisense DNA complement of flt1 mRNA abolished the hypoxia-induced stimulation of pericyte growth. Finally, exogenous VEGF stimulated the migration of pericytes in a dose-dependent manner. The results thus suggest that VEGF, which has been thought to be a specific mitogen for EC, also acts on neighboring pericytes, probably in both autocrine and paracrine manners, and that the hypoxia-induced overproduction of VEGF could promote not only EC sprouting but also the recruitment of pericytes, thereby contributing to the maturation of newly formed microvessels.
Assuntos
Fatores de Crescimento Endotelial/farmacologia , Fatores de Crescimento Endotelial/fisiologia , Linfocinas/farmacologia , Linfocinas/fisiologia , Pericitos/citologia , Animais , Bovinos , Divisão Celular/efeitos dos fármacos , Hipóxia Celular , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Quimiotaxia/fisiologia , DNA/biossíntese , Primers do DNA , Fatores de Crescimento Endotelial/genética , Regulação da Expressão Gênica , Linfocinas/genética , Neovascularização Fisiológica , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Sondas de Oligonucleotídeos , Pericitos/efeitos dos fármacos , Pericitos/fisiologia , Receptores Proteína Tirosina Quinases/genética , Receptores de Fatores de Crescimento/genética , Receptores de Fatores de Crescimento do Endotélio Vascular , Vasos Retinianos/citologia , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Several thrombogenic abnormalities are associated with diabetes. To investigate the underlying molecular mechanisms, we examined the effects of advanced glycation endproducts (AGE), non-enzymatically glycated protein derivatives, on the production of prostacyclin (PGI2), an anti-thrombogenic prostanoid, and of plasminogen activator inhibitor-1 (PAI-1), a fast-acting serine protease inhibitor of fibrinolysis, in human microvascular endothelial cells (EC). Firstly, AGE-bovine serum albumin (BSA) but not non-glycated BSA, was found to considerably decrease the production of PGI2 to about two-thirds of the control value. Secondly, quantitative reverse transcription-polymerase chain reaction showed that AGE-BSA increased the EC levels of mRNA coding for PAI-1, this being associated with a concomitant increase in the immunoreactive PAI-1 contents and the anti-fibrinolytic activity. Thirdly, the effects of AGE on PGI2 and PAI-1 syntheses in EC were found to be mediated by a receptor for AGE (RAGE) because antisense DNA against RAGE mRNA could reverse the AGE effects. Further, it was found that AGE decreased the intracellular cyclic AMP concentrations in EC and that cyclic AMP agonists such as dibutyryl cyclic AMP, forskolin and PGI2 analogue reduced the AGE-stimulated PAI-1 production, suggesting the involvement of cyclic AMP in the AGE-signalling pathway. The results thus suggest that AGE have the ability to cause platelet aggregation and fibrin stabilization, resulting in a predisposition to thrombogenesis and thereby contributing to the development and progression of diabetic vascular complications.