RESUMO
We describe investigations to expand the scope of next generation maleimide cross-linkers for the construction of homogeneous protein-protein conjugates. Diiodomaleimides are shown to offer the ideal properties of rapid bioconjugation with reduced hydrolysis, allowing the cross-linking of even sterically hindered systems. The optimized linkers are exploited to link human serum albumin to antibody fragments (Fab or scFv) as a prospective half-life extension platform, with retention of antigen binding and robust serum stability. Finally, a triprotein conjugate is formed, by linking scFv antibody fragments targeting carcinoembryonic antigen. This tri-scFv is shown to infer a combination of greater antigen avidity and increased in vivo half-life, representing a promising platform for antibody therapeutic development.
Assuntos
Reagentes de Ligações Cruzadas/química , Imunoconjugados/química , Maleimidas/química , Albumina Sérica Humana/química , Anticorpos de Cadeia Única/química , Humanos , Hidrólise , Fragmentos Fab das Imunoglobulinas/química , Modelos MolecularesRESUMO
BACKGROUND: Non-invasive measures of tumour vascular perfusion are desirable, in order to assess response to vascular targeting (or modifying) therapies. In this study, hepatic arterial spin labelling (ASL) magnetic resonance imaging (MRI) was investigated to measure acute changes in perfusion of colorectal cancer in the liver, in response to vascular disruption therapy with OXi4503. METHODS: SW1222 and LS174T tumours were established in the liver of MF1 nu/nu mice via intrasplenic injection. Perfusion and R2(*) MRI measurements were acquired with an Agilent 9.4T horizontal bore scanner, before and at 90 min after 40 mg kg(-1) OXi4503. RESULTS: A significant decrease in SW1222 tumour perfusion was observed (-43±33%, P<0.005). LS174T tumours had a significantly lower baseline level of perfusion. Intrinsic susceptibility MRI showed a significant increase in R2(*) in LS174T tumours (28±25%, P<0.05). An association was found between the change in tumour perfusion and the proximity to large vessels, with pre-treatment blood flow predictive of subsequent response. Histological evaluation confirmed the onset of necrosis and evidence of heterogeneous response between tumour deposits. CONCLUSIONS: Hepatic ASL-MRI can detect acute response to targeted tumour vascular disruption entirely non-invasively. Hepatic ASL of liver tumours has potential for use in a clinical setting.
Assuntos
Artéria Hepática/patologia , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/patologia , Fígado/patologia , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Feminino , Angiografia por Ressonância Magnética/métodos , Espectroscopia de Ressonância Magnética/métodos , Camundongos , Camundongos Nus , Marcadores de SpinRESUMO
Ultrasound-mediated drug delivery is a promising means of enhancing delivery, distribution and effectiveness of drugs within tumours. In this review, prospects for exploiting ultrasound to improve the tumour delivery and distribution of radiolabelled antibodies for radioimmunotherapy and to overcome barriers imposed by tumour microenvironment are discussed.
Assuntos
Terapia de Alvo Molecular/métodos , Neoplasias/diagnóstico por imagem , Neoplasias/radioterapia , Radioimunoterapia/métodos , Animais , Anticorpos/uso terapêutico , Humanos , UltrassonografiaRESUMO
Nuclear imaging in conjunction with radioactive tracers enables noninvasive measurements of biochemical events in vivo. However, access to tracers remains limited due to the lack of methods for rapid assembly of radiolabeled molecules with the prerequisite biological activity. Herein, we report a one-pot, three-component, copper(II)-mediated reaction of azides, alkynes, and [(125)I]iodide to yield 5-[(125)I]iodo-1,2,3-triazoles. Using a selection of azides and alkynes in a combinatorial approach, we have synthesized a library of structurally diverse (125)I-labeled triazoles functionalized with bioconjugation groups, fluorescent dyes, and biomolecules. Our preliminary biological evaluation suggests that 5-[(125)I]iodo-1,2,3-triazoles are resistant to deiodination in vivo, both as small molecular probes and as antibody conjugates. The ability to incorporate radioactive iodide into triazoles directly from the parent azides and alkynes makes the method broadly applicable and offers the potential to rapidly assemble molecular probes from an array of structurally diverse, and readily available, building blocks.
Assuntos
Radioisótopos do Iodo/química , Sondas Moleculares/química , Triazóis/química , Sondas Moleculares/síntese química , Estrutura Molecular , Radioquímica , Fatores de Tempo , Triazóis/síntese químicaRESUMO
The potential of radioimmunotherapy to selectively kill tumour cells is well established. However, optimisation is required with regards to increasing tumour localisation of antibodies. We used the PDGF-receptor inhibitor imatinib mesylate to improve tumour-specific antibody localisation in two models of colorectal adenocarcinoma and correlated antibody localisation with changes to tumour microvasculature. Mice bearing human colorectal xenografts (LS174T or SW1222) were treated with imatinib prior to administration of radiolabeled anti-CEA antibodies ((125)I-A5B7). Whole tumour and regional localisation of radiolabeled antibodies were measured. Microvessel density and pericyte coverage were quantified in whole tumours and correlated with (125)I-A5B7 localisation. Imatinib increased uptake of (125)I-A5B7 in LS174T but not SW1222 tumours after 48 h (p < 0.05). Imatinib reduced microvessel density in both models (p < 0.05) but reduced pericyte attachment to endothelial cells only in SW1222 xenografts (p < 0.05). Imatinib increases antibody distribution in LS174T tumours but not SW1222 tumours, and this correlated to changes in tumour microvessels. Accelerated clearance of radiolabeled antibody from normal tissues in both models resulted in enhanced tumour to normal tissue ratios. This improvement in tumour/normal tissue ratio has potential clinical benefit from a therapy and imaging perspective, and merits further investigation.
Assuntos
Adenocarcinoma/terapia , Anticorpos Monoclonais/uso terapêutico , Neoplasias Colorretais/terapia , Modelos Animais de Doenças , Piperazinas/uso terapêutico , Pirimidinas/uso terapêutico , Radioimunoterapia , Receptor beta de Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Adenocarcinoma/imunologia , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Anticorpos Monoclonais/farmacocinética , Antineoplásicos/uso terapêutico , Benzamidas , Antígeno Carcinoembrionário/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Terapia Combinada , Feminino , Imunofluorescência , Humanos , Mesilato de Imatinib , Técnicas Imunoenzimáticas , Camundongos , Camundongos Nus , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Distribuição Tecidual , Transplante HeterólogoRESUMO
Induction of apoptosis is often necessary for successful cancer therapy, and the non-invasive monitoring of apoptosis post-therapy could assist in clinical decision making. Isatins are a class of compounds that target activated caspase-3 during apoptosis. Here we report the synthesis of the 5-iodo-1,2,3-triazole (FITI) analog of the PET tracer [18F]ICMT11 as a candidate tracer for imaging of apoptosis with SPECT, as well as PET. Labelling with radioiodine (123,125I) was achieved in 55 ± 12% radiochemical yield through a chelator-accelerated one-pot cycloaddition reaction mediated by copper(I) catalysis. The caspase-3 binding affinity and selectivity of FITI compares favourably to that of [18F]ICMT11 (Ki = 6.1 ± 0.9 nM and 12.4 ± 4.7 nM, respectively). In biodistribution studies, etoposide-induced cell death in a SW1222 xenograft model resulted in a 2-fold increase in tumour uptake of the tracer. However, the tumour uptake was too low to allow in vivo imaging of apoptosis with SPECT.
Assuntos
Apoptose/efeitos dos fármacos , Caspase 3/isolamento & purificação , Radioisótopos do Iodo/farmacologia , Neoplasias/diagnóstico por imagem , Animais , Apoptose/genética , Caspase 3/química , Caspase 3/genética , Linhagem Celular Tumoral , Cobre/química , Radioisótopos de Flúor/química , Radioisótopos de Flúor/farmacologia , Xenoenxertos , Humanos , Radioisótopos do Iodo/química , Isatina/síntese química , Isatina/farmacologia , Camundongos , Neoplasias/patologia , Neoplasias/terapia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacologia , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Triazóis/síntese química , Triazóis/farmacologiaRESUMO
The combination of early diagnosis and complete surgical resection offers the greatest prospect of curative cancer treatment. An iodine-124/fluorescein-based dual-modality labeling reagent, 124I-Green, constitutes a generic tool for one-step installation of a positron emission tomography (PET) and a fluorescent reporter to any cancer-specific antibody. The resulting antibody conjugate would allow both cancer PET imaging and intraoperative fluorescence-guided surgery. 124I-Green was synthesized in excellent radiochemical yields of 92 ± 5% (n = 4) determined by HPLC with an improved one-pot three-component radioiodination reaction. The A5B7 carcinoembryonic antigen (CEA)-specific antibody was conjugated to 124I-Green. High tumor uptake of the dual-labeled A5B7 of 20.21 ± 2.70, 13.31 ± 0.73, and 10.64 ± 1.86%ID/g was observed in CEA-expressing SW1222 xenograft mouse model (n = 3) at 24, 48, and 72 h post intravenous injection, respectively. The xenografts were clearly visualized by both PET/CT and ex vivo fluorescence imaging. These encouraging results warrant the further translational development of 124I-Green for cancer PET imaging and fluorescence-guided surgery.
Assuntos
Imunoconjugados/química , Radioisótopos do Iodo/química , Neoplasias/diagnóstico por imagem , Neoplasias/cirurgia , Animais , Anticorpos Monoclonais/química , Xenoenxertos , Humanos , Camundongos , Imagem Óptica , Tomografia por Emissão de Pósitrons/métodosRESUMO
The magnetic properties and safety of dextran-coated superparamagnetic iron oxide nanoparticles (SPIONs) have facilitated their clinical use as MRI contrast agents and stimulated research on applications for SPIONs in particle imaging and magnetic hyperthermia. The wider clinical potential of SPIONs, however, has been limited by their rapid removal from circulation via the reticuloendothelial system (RES). We explored the possibility of extending SPION circulatory time using fucoidan, a seaweed-derived food supplement, to inhibit RES uptake. The effects of fucoidan on SPION biodistribution were evaluated using ferucarbotran, which in its pharmaceutical formulation (Resovist) targets the RES. Ferucarbotran was radiolabeled at the iron oxide core with technetium-99m (99mTc; t1/2 = 6 h) or zirconium-89 (89Zr; t1/2 = 3.3 days). Results obtained with 99mTc-ferucarbotran demonstrated that administration of fucoidan led to a 4-fold increase in the circulatory half-life (t1/2 slow) from 37.4 to 150 min (n = 4; P < 0.0001). To investigate whether a longer circulatory half-life could lead to concomitant increased tumor uptake, the effects of fucoidan were tested with 89Zr-ferucarbotran in mice bearing syngeneic subcutaneous (GL261) tumors. In this model, the longer circulatory half-life achieved with fucoidan was associated with a doubling in tumor SPION uptake (n = 5; P < 0.001). Fucoidan was also effective in significantly increasing the circulatory half-life of perimag-COOH, a commercially available SPION with a larger hydrodynamic size (130 nm) than ferucarbotran (65 nm). These findings indicate successful diversion of SPIONs away from the hepatic RES and show realistic potential for future clinical applications.
Assuntos
Anticorpos Monoclonais/análise , Química Click , Radioisótopos de Índio/química , Neoplasias/diagnóstico , Triazóis/química , Animais , Anticorpos Monoclonais/química , Antígeno Carcinoembrionário/análise , Química Click/métodos , Cobre/química , Diagnóstico por Imagem/métodos , Fluorescência , Humanos , CamundongosRESUMO
The SMAD-mediated induction of connective tissue growth factor (CTGF), a fibroproliferative cytokine, by transforming growth factor (TGF)beta is required for the development of sustained fibrosis in humans. Here, we show that in fibroblasts, activation of the Ras/MEK/ERK pathway is required for the SMAD-mediated induction of CTGF by TGFbeta2. We then show that activation of protein kinase A (PKA) in fibroblasts is able to block Ras/MEK/ERK signaling and abolish the fibrotic response. Previously, we found that prostacyclin agonists were able to prevent the induction of CTGF in fibroblasts, and in patients with the fibrotic disease scleroderma. Here, we confirm the in vitro and in vivo antifibrotic effects of prostacyclin derivatives and show that these effects are due to PKA-dependent inhibition of the Ras/MEK/ERK pathway. Ras/MEK/ERK does not directly affect SMAD signaling. The coordinate and varied biological responses to TGFbeta are in part due to the interactions of signaling pathways within target cells. Specific inhibition of fibroblast Ras/MEK/ERK signaling might prevent fibrosis while leaving other physiological effects of TGFbeta unaltered.
Assuntos
Iloprosta/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Transformador beta/antagonistas & inibidores , Animais , Fator de Crescimento do Tecido Conjuntivo , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Fibrose/prevenção & controle , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Peptídeos e Proteínas de Sinalização Intercelular/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Modelos Biológicos , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , RNA Mensageiro/biossíntese , Ratos , Proteínas Smad , Transativadores/metabolismoRESUMO
Herein we report the use of next generation maleimides (NGMs) for the construction of a potent antibody-drug conjugate (ADC) via functional disulfide bridging. The linker has excellent stability in blood serum and the ADC, armed with monomethyl auristatin E (MMAE), shows excellent potency and cancer cell selectivity in vitro.
Assuntos
Anticorpos Monoclonais/química , Dissulfetos/química , Imunoconjugados/química , Oligopeptídeos/química , Proliferação de Células/efeitos dos fármacos , Química Click , Corantes Fluorescentes/química , Humanos , Imunoconjugados/toxicidade , Células MCF-7 , Trastuzumab/químicaRESUMO
UNLABELLED: Despite extensive efforts to improve the clinical management of patients with colorectal cancer, approved treatments for advanced disease offer limited survival benefit. Therefore, the identification of novel treatment strategies is essential. We evaluated the preclinical efficacy of combination radioimmunotherapy, using a humanized (131)I-labeled anti-carcinoembryonic antigen antibody ((131)I-huA5B7), with cetuximab in colorectal cancer (CRC). METHODS: Three human CRC cell lines--SW1222, LoVo, and LS174T--were used to generate subcutaneous xenografts, and stably luciferase-transfected SW1222 cells were used to establish a model of hepatic metastases in immunocompromised mice. Imaging and biodistribution studies were conducted to confirm the selective tumor localization of (131)I-huA5B7. Efficacy was evaluated on the basis of tumor growth delay and survival, along with markers of DNA damage response, cell cycle, proliferation, and apoptosis. RESULTS: Selective tumor targeting was achieved with (131)I-huA5B7 alone or in combination with cetuximab without observable toxicity. Compared with monotherapy, combining cetuximab with radioimmunotherapy significantly and synergistically reduced tumor growth and prolonged survival of mice in 2 of the subcutaneous and in the metastatic tumor model. Evidence of DNA damage, G2/M arrest, significantly decreased proliferation, and increased apoptosis were observed with radioimmunotherapy and the combination therapy. However, a significant decrease in DNA-protein kinase expression with the combination regimen suggests that the addition of cetuximab suppressed DNA repair. CONCLUSION: Our results demonstrate enhanced therapeutic efficacy with the combination of cetuximab and radioimmunotherapy in CRC, which could potentially translate into successful clinical outcomes. This strategy could improve the treatment of residual disease postoperatively and ultimately prevent or delay recurrence. Furthermore, other carcinoembryonic antigen-expressing malignancies could also benefit from this approach.
Assuntos
Anticorpos Monoclonais Humanizados/administração & dosagem , Anticorpos Monoclonais/química , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/radioterapia , Radioimunoterapia/métodos , Animais , Apoptose , Antígeno Carcinoembrionário/metabolismo , Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Cetuximab , Neoplasias Colorretais/diagnóstico por imagem , Dano ao DNA , Feminino , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Nus , Camundongos SCID , Metástase Neoplásica , Cintilografia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The vascular network is a series of linked conduits of blood vessels composed of the endothelium, a monolayer of cells that adorn the vessel lumen and surrounding layer(s) of mesenchymal cells (vascular smooth muscle, pericytes and fibroblasts). In addition to providing structural support, the mesenchymal cells are essential for vessel contractility. The extracellular matrix is a major constituent of blood vessels and provides a framework in which these various cell types are attached and embedded. The composition and organization of vascular extracellular matrix is primarily controlled by the mesenchymal cells, and is also responsible for the mechanical properties of the vessel wall, forming complex networks of structural proteins which are highly regulated. The extracellular matrix also plays a central role in cellular adhesion, differentiation and proliferation. This review examines the cellular and extracellular matrix components of vessels, with specific emphasis on the regulation of collagen type I and implications in vascular disease.
Assuntos
Vasos Sanguíneos/metabolismo , Matriz Extracelular/metabolismo , Mesoderma/metabolismo , Animais , Vasos Sanguíneos/citologia , Colágeno Tipo I/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Humanos , Mesoderma/citologia , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Pericitos/citologia , Pericitos/metabolismo , Doenças Vasculares/metabolismo , Doenças Vasculares/fisiopatologiaRESUMO
There is a need for biomarkers that are useful for noninvasive imaging of tumor pathophysiology and drug efficacy. Through its use of endogenous water, diffusion-weighted MRI (DW-MRI) can be used to probe local tissue architecture and structure. However, most DW-MRI studies of cancer tissues have relied on simplistic mathematical models, such as apparent diffusion coefficient (ADC) or intravoxel incoherent motion (IVIM) models, which produce equivocal results on the relation of the model parameter estimate with the underlying tissue microstructure. Here, we present a novel technique called VERDICT (Vascular, Extracellular and Restricted Diffusion for Cytometry in Tumors) to quantify and map histologic features of tumors in vivo. VERDICT couples DW-MRI to a mathematical model of tumor tissue to access features such as cell size, vascular volume fraction, intra- and extracellular volume fractions, and pseudo-diffusivity associated with blood flow. To illustrate VERDICT, we used two tumor xenograft models of colorectal cancer with different cellular and vascular phenotypes. Our experiments visualized known differences in the tissue microstructure of each model and the significant decrease in cell volume resulting from administration of the cytotoxic drug gemcitabine, reflecting the apoptotic volume decrease. In contrast, the standard ADC and IVIM models failed to detect either of these differences. Our results illustrate the superior features of VERDICT for cancer imaging, establishing it as a noninvasive method to monitor and stratify treatment responses.
Assuntos
Imagem de Difusão por Ressonância Magnética/métodos , Neoplasias/ultraestrutura , Animais , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/uso terapêutico , Feminino , Humanos , Camundongos , Modelos Teóricos , Neoplasias/irrigação sanguínea , Neoplasias/tratamento farmacológico , GencitabinaRESUMO
Tumors have a greater reliance on anaerobic glycolysis for energy production than normal tissues. We developed a noninvasive method for imaging glucose uptake in vivo that is based on magnetic resonance imaging and allows the uptake of unlabeled glucose to be measured through the chemical exchange of protons between hydroxyl groups and water. This method differs from existing molecular imaging methods because it permits detection of the delivery and uptake of a metabolically active compound in physiological quantities. We show that our technique, named glucose chemical exchange saturation transfer (glucoCEST), is sensitive to tumor glucose accumulation in colorectal tumor models and can distinguish tumor types with differing metabolic characteristics and pathophysiologies. The results of this study suggest that glucoCEST has potential as a useful and cost-effective method for characterizing disease and assessing response to therapy in the clinic.
Assuntos
Glucose/metabolismo , Imageamento por Ressonância Magnética , Neoplasias/metabolismo , Aminoácidos/metabolismo , Animais , Autorradiografia , Isótopos de Carbono , Hipóxia Celular , Feminino , Fluordesoxiglucose F18 , Glucose/química , Humanos , Camundongos , Microscopia de Fluorescência , Neoplasias/patologia , Perfusão , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Radioimmunotherapy (RIT) is a cancer treatment that exploits the specific targeting capability of monoclonal antibodies to deliver cytotoxic radionuclides to antigen-expressing tumor cells or stromal targets. While this has been extremely successful in the treatment of hematologic malignancies, RIT of solid tumors has produced less prolonged effects. In our laboratory, we have developed a bench-to-bedside translational pipeline with the aim of optimizing RIT for solid tumors. We will show how preclinical models of colorectal adenocarcinoma were initially used to study reciprocal interactions between elements of the tumor microenvironment and RIT and to test novel therapeutic strategies. These studies were then used to facilitate the design of novel trials carried out in close collaboration with our clinical colleagues.
Assuntos
Neoplasias Colorretais/radioterapia , Radioimunoterapia/métodos , Humanos , Projetos de Pesquisa , Microambiente TumoralRESUMO
By providing a source of alpha-smooth muscle actin (alpha-SMA)-expressing myofibroblasts, microvascular pericytes contribute to the matrix remodeling that occurs during tissue repair. However, the extent to which pericytes may contribute to the fibroblast phenotype post-repair is unknown. In this report, we test whether pericytes isolated from human placenta can in principle become fibroblast-like. Pericytes were cultured in vitro for 11 passages. The Affymetrix mRNA expression profile of passage 2 and passage 11 pericytes was compared. The expression of type I collagen, thrombospondin and fibronectin mRNAs was induced by passaging pericytes in culture. This induction of a fibroblast phenotype was paralleled by induction of connective tissue growth factor (CTGF/CCN2) and type I collagen protein expression and the fibroblast marker ASO2. These results indicate that, in principle, pericytes have the capacity to become fibroblast-like and that pericytes may contribute to the population of fibroblasts in a healed wound.
RESUMO
OBJECTIVE: Connective tissue growth factor (CTGF; CCN2) is overexpressed in systemic sclerosis (SSc) and has been hypothesized to be a key mediator of the pulmonary fibrosis frequently observed in this disease. CTGF is induced by transforming growth factor beta (TGFbeta) and is a mediator of some profibrotic effects of TGFbeta in vitro. This study was undertaken to investigate the role of CTGF in enhanced expression of type I collagen in bleomycin-induced lung fibrosis, and to delineate the mechanisms of action underlying the effects of CTGF on Col1a2 (collagen gene type I alpha2) in this mouse model and in human pulmonary fibroblasts. METHODS: Transgenic mice that were carrying luciferase and beta-galactosidase reporter genes driven by the Col1a2 enhancer/promoter and the CTGF promoter, respectively, were injected with bleomycin to induce lung fibrosis (or saline as control), and the extracted pulmonary fibroblasts were incubated with CTGF blocking agents. In vitro, transient transfection, promoter/reporter constructs, and electrophoretic mobility shift assays were used to determine the mechanisms of action of CTGF in pulmonary fibroblasts. RESULTS: In the mouse lung tissue, CTGF expression and promoter activity peaked 1 week after bleomycin challenge, whereas type I collagen expression and Col1a2 promoter activity peaked 2 weeks postchallenge. Fibroblasts isolated from the mouse lungs 14 days after bleomycin treatment retained a profibrotic expression pattern, characterized by greatly elevated levels of type I collagen and CTGF protein and increased promoter activity. In vitro, inhibition of CTGF by specific small interfering RNA and neutralizing antibodies reduced the collagen protein expression and Col1a2 promoter activity. Moreover, in vivo, anti-CTGF antibodies applied after bleomycin challenge significantly reduced the Col1a2 promoter activity by approximately 25%. The enhanced Col1a2 promoter activity in fibroblasts from bleomycin-treated lungs was partly dependent on Smad signaling, whereas CTGF acted on the Col1a2 promoter by a mechanism that was independent of the Smad binding site, but was, instead, dependent on the ERK-1/2 and JNK MAPK pathways. The CTGF effect was mapped to the proximal promoter region surrounding the inverted CCAAT box, possibly involving CREB and c-Jun. In human lung fibroblasts, the human COL1A2 promoter responded in a similar manner, and the mechanisms of action also involved ERK-1/2 and JNK signaling. CONCLUSION: Our results clearly define a direct profibrotic effect of CTGF and demonstrate its contribution to lung fibrosis through transcriptional activation of Col1a2. Blocking strategies revealed the signaling mechanisms involved. These findings show CTGF to be a rational target for therapy in fibrotic diseases such as SSc.
Assuntos
Fator de Crescimento do Tecido Conjuntivo/metabolismo , Fibroblastos/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fibrose Pulmonar/metabolismo , Transcrição Gênica/fisiologia , Animais , Sequência de Bases , Bleomicina/efeitos adversos , Células Cultivadas , Colágeno/metabolismo , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Fator de Crescimento do Tecido Conjuntivo/antagonistas & inibidores , Fator de Crescimento do Tecido Conjuntivo/genética , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Humanos , MAP Quinase Quinase 4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-jun/metabolismo , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/patologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/fisiologia , Proteínas Smad/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
The concept of mesenchymal fibroblasts has evolved over the past two decades from a relatively inert structural cell type to a dynamic, pluripotent cell lineage controlling normal connective tissue formation, homeostasis, and repair and as principle players in pathogenic scarring and fibrosis. In wound healing and tissue repair, fibroblasts provide proinflammatory signals and synthesize interstitial collagens, fibronectins, and other matrix components to repair the damaged tissue. Fibroblasts can differentiate into the myofibroblast, a specialized contractile cell type responsible for wound closure, tissue contraction, and scarring. This article reviews our current understanding of the origins of mesenchymal cells and their role in excessive scarring and fibrogenesis and in the systemic fibrotic disease scleroderma.
Assuntos
Fibroblastos/patologia , Escleroderma Sistêmico/patologia , Diferenciação Celular , Fibrose/patologia , Humanos , Células-Tronco Mesenquimais/citologia , Escleroderma Sistêmico/etiologia , Transdução de SinaisRESUMO
OBJECTIVE: The potent physiologic endogenous angiogenic response to ischemic stimuli is often suboptimal, and therefore, a better understanding of the basic mechanisms is essential for the use in therapeutic angiogenesis. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor that promotes ischemia-driven angiogenesis and is induced when the HIF-1alpha subunit is upregulated. However, little is known about the endogenous angiogenic response and the role of HIF-1alpha in human critical limb ischemia (CLI). We aimed to investigate the extent of the angiogenic response and the expression of HIF-1alpha in the lower limbs of CLI patients. METHOD: Skeletal muscle biopsy specimens were obtained from the lower limbs of 12 patients with CLI and 12 patients without limb ischemia (controls), with ethical committee approval. Microvessel density (MVD) was determined by using endothelial marker anti-CD31, and HIF-1alpha expressions were determined by immunohistochemistry. MVD was measured as the median number of microvessels in x200 magnification fields. Five random fields per section and three sections per biopsy specimen were analyzed. Enzyme-linked immunoabsorbent assay and Western blotting were used to quantify the HIF-1alpha levels. Colocalization between cell-specific antigens was investigated by double immunofluorescence labelling by using confocal microscopy. Statistical analyses were performed with the Mann-Whitney U test. RESULTS: The CLI group have significantly higher MVD, with an increase of 2.7-fold compared with the controls (P < 0.001). HIF-1alpha expression was significantly increased in CLI muscles (P < 0.001) and was localized to vascular endothelial cells. CONCLUSIONS: Our findings suggest that the endogenous angiogenic response occurs in CLI. The increased HIF-1alpha level and colocalization to vascular endothelial cells suggest that HIF-1alpha plays a role in the physiologic endogenous angiogenic response in CLI. Therefore, augmentation of the HIF-1alpha pathway may be an important aspect in therapeutic angiogenesis.