RESUMO
Oncolytic adenovirus-mediated gene therapy shows promise for cancer treatment; however, the systemic delivery of oncolytic adenovirus to tumors remains challenging. Recently, mesenchymal stem cells (MSCs) have emerged as potential vehicles for improving delivery. Yet, because the oncolytic adenovirus replicates in MSCs, balancing MSC viability with viral load is key to achieving optimal therapeutic effect. We thus developed an all-in-one Tet-on system that can regulate replication of oncolytic adenovirus. Then, we loaded the novel oncolytic adenovirus carrying interleukin (IL)-24 and/or Endostatin in human umbilical cord blood-mesenchymal stem cells (hUCB-MSCs) for glioma therapy. In vitro assays demonstrated that this novel oncolytic adenovirus could efficiently replicate and kill glioma cells while sparing normal cells. Moreover, doxycycline effectively regulated oncolytic adenovirus replication in the hUCB-MSCs. The doxycycline induction group with dual expression of IL-24 and Endostatin exhibited significantly greater antitumor effects than other groups in a xenograft model of glioma. Thus, this strategy for systemic delivery of oncolytic adenovirus with its oncolytic activity controlled by a Tet-on system is a promising method for achieving antitumor efficacy in glioma, especially for metastatic tumors.
Assuntos
Neoplasias Encefálicas/terapia , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Endostatinas/biossíntese , Terapia Genética , Glioma/terapia , Interleucinas/biossíntese , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/virologia , Terapia Viral Oncolítica , Vírus Oncolíticos/genética , Animais , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/virologia , Morte Celular , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Endostatinas/genética , Feminino , Vetores Genéticos , Glioma/genética , Glioma/metabolismo , Glioma/virologia , Humanos , Interleucinas/genética , Camundongos Endogâmicos BALB C , Camundongos Nus , Vírus Oncolíticos/crescimento & desenvolvimento , Carga Tumoral , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Anti-CD105 mAb-conjugated immunoliposomes, loaded with secreted mouse endostatin gene, were developed for targeted tumor imaging and antiangiogenic gene therapy. METHODS: The liposomes were investigated for size, zeta-potential, lipid content, antibody binding ability, and pcDNA loading capacity. The ability of immunoliposomes to target tumor-derived endothelial cells and perform gene transfer in vitro was measured and their basic biocompatibility was evaluated. A nude mouse/breast cancer xenograft model was used to examine the tumor internalization of fluorescent-labeled liposomes and the clinical potential of immnuoliposomes loaded with pcDNA3.1-CSF1-endostatin. RESULTS: Loaded immunoliposomes were homogenously distributed with a well-defined spherical shape and bilayer, diameter of 122 ± 11 nm, and zeta potential + 1.40 mV. No significant differences were observed in body weight, liver index, oxidative stress, or liver and kidney function in mice after liposomes exposure. The addition of CD105 mAb to liposomes conferred the ability to target tumor-derived endothelial cells in vitro and in vivo. Systemic intravenous administration of fluorescent immunoliposomes in the xenograft model resulted in selective and efficient internalization in tumor vasculature. Treatment of mice with pcDNA3.1-CSF1-endostatin-loaded immunoliposomes suppressed tumor growth by 71%. CONCLUSIONS: These data demonstrate the advantages of using anti-CD105 mAb-conjugated immunoliposomes to enhance tumor targeting, imaging, and gene transfer applications.
Assuntos
Anticorpos Monoclonais/administração & dosagem , Endoglina/antagonistas & inibidores , Endostatinas/genética , Lipossomos , Imagem Molecular , Neoplasias/diagnóstico , Neoplasias/genética , Animais , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Endostatinas/biossíntese , Feminino , Técnicas de Transferência de Genes , Terapia Genética , Glutationa/metabolismo , Humanos , Lipossomos/química , Lipossomos/ultraestrutura , Camundongos , Neoplasias/terapia , Imagem Óptica/métodos , Plasmídeos/química , Plasmídeos/genética , Superóxido Dismutase/metabolismo , Espectrometria de Massas em Tandem , TransgenesRESUMO
Hirudin's ability to increase angiogenesis in ischemic flap tissue and improve the flaps survival has been demonstrated in our previous studies. However, the knowledge about hirudin functional role in angiogenesis is still limited. In the present study, we investigate the effects of locally injected hirudin on the expression of VEGF, endostatin and thrombospondin-1 (TSP-1) using rat model. Caudally based dorsal skin flaps were created and were treated with hirudin or normal saline. Result showed that the flap survival was improved by hirudin treatment relative to the control. Treatment of flaps with hirudin exerted significant angiogenic effect as evidenced by increased VEGF expression and reduced endostatin and TSP-1 production (p<0.01), and promoted neovascularization (microvascular density, p<0.01). Moreover, hirudin treatment increased the ERK1/2 phosphorylation, while attenuated the phosphorylation of p38 MAPK, and the addition of thrombin could reverse these effects of hirudin on ERK1/2 and p38 MAPK activity. The MEK inhibitor blocked the hirudin-induced VEGF expression, and the p38 MAPK inhibitor attenuated the thrombin-induced TSP-1 expression. Furthermore, a specific inhibitor of p38 MAPK activates ERK1/2 in ischemic flaps, suggesting that cross-talk between p38 MAPK and ERK might exist in rat ischemic flap tissue. Moreover, either the hirudin or SCH79797 (PAR1 antagonist) could attenuate the p38 MAPK phosphorylation and increases the ERK1/2 phosphorylation, indicating that the cross-talk between p38 MAPK and ERK1/2 modulated by thrombin/PAR1 signaling may participate in the process of hirudin-stimulated ERK1/2 signaling. In conclusion, these observations suggest that hirudin exerts its angiogenesis effect by regulating the expression of angiogenic and antiangiogenic factors via a cross-talk of p38 MAPK-ERK pathway.
Assuntos
Endostatinas/biossíntese , Hirudinas/administração & dosagem , Trombospondinas/biossíntese , Fator A de Crescimento do Endotélio Vascular/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Animais , Endostatinas/genética , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Retalho Miocutâneo/patologia , Neovascularização Fisiológica/efeitos dos fármacos , Neovascularização Fisiológica/genética , Fosforilação/efeitos dos fármacos , Pirróis/administração & dosagem , Quinazolinas/administração & dosagem , Ratos , Pele/efeitos dos fármacos , Pele/patologia , Trombospondinas/genética , Fator A de Crescimento do Endotélio Vascular/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
To develop optimal therapeutics is one of the hotspots in both clinical and basic melanoma studies. Previous studies indicate that fibroblast growth factors (b-FGF/FGF-2), an angiogenesis inducer beyond VEGF, might be a potential drug target in melanoma. As a novel anti-angiogenesis peptide drug, Endostar has shown promising therapeutic efficacy in non-small cell lung cancer. However, the effect of Endostar on b-FGF-induced angiogenesis in melanoma is unraveled. To this end, both in vivo and in vitro experiments were conducted and it was found that treatment of Endostar could inhibit tumor growth, which was accompanied by decreased micro-vessel density and serum b-FGF levels in a mouse melanoma model. In addition, treatment with Endostar in blood vessel endothelial cells could reduce their proliferation, cell migration and tube formation capacity in a dosage-dependent manner. Moreover, treatment of Endostar could also attenuate b-FGF-activated phosphorylation of p38 and ERK1/2 in HUVECs. These findings indicate that Endostar might exert its anti-tumor effect via suppressing b-FGF-induced angiogenesis and b-FGF-activated MAPK signaling pathway, suggesting that Endostar might be a potential choice for clinical melanoma treatment.
Assuntos
Inibidores da Angiogênese/farmacologia , Endostatinas/farmacologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/tratamento farmacológico , Neovascularização Patológica , Neovascularização Fisiológica/efeitos dos fármacos , Neoplasias Cutâneas/irrigação sanguínea , Neoplasias Cutâneas/tratamento farmacológico , Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/genética , Animais , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Endostatinas/biossíntese , Endostatinas/genética , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosforilação , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Neoplasias Cutâneas/patologia , Carga Tumoral/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
MSCs-based therapy for cancer is a relatively new but rapidly growing area of research. Human term placenta, an attractive source of MSCs (PMSCs), appears to have great advantage due to its easy access without invasive procedures, its lack of ethical issues and its high-throughput and young age. In the present study, we isolated MSCs from placenta and characterized their morphology and differentiation capacities. We next investigated the underlying antitumor effects and the potential mechanism of PMSCs to express endostatin using adenoviral transduction (Ad-Endo) in a colorectal peritoneal carcinomatosis (CRPC) mouse model. For in vitro experiments, the migratory potential of Ad-Endo-PMSCs towards tumor cells was demonstrated using a double-chamber assay, and the anti-angiogenesis ability of endostatin from engineered PMSCs was evaluated using the tube formation assay. For the in vivo study, mice harboring CT26 colorectal cancer indicated a significant reduction in tumor nodules and a prolongation of survival following Ad-Endo-PMSCs therapy. These observations were associated with significantly decreased tumor cell proliferation and blood vessel counts as well as increased tumor cell apoptosis. These data suggested the potential of PMSCs-based gene therapy for the targeted delivery of therapeutic proteins in cancer.
Assuntos
Carcinogênese/genética , Neoplasias Colorretais/genética , Endostatinas/biossíntese , Peritônio/patologia , Adenoviridae , Animais , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica , Terapia Genética , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Camundongos , Placenta/citologia , GravidezRESUMO
Viruses have demonstrated strong potential for the therapeutic targeting of glioblastoma stem cells (GSCs). In this study, the use of a herpes simplex virus carrying endostatin-angiostatin (VAE) as a novel therapeutic targeting strategy for glioblastoma-derived cancer stem cells was investigated. We isolated six stable GSC-enriched cultures from 36 human glioblastoma specimens and selected one of the stable GSCs lines for establishing GSC-carrying orthotopic nude mouse models. The following results were obtained: (a) VAE rapidly proliferated in GSCs and expressed endo-angio in vitro and in vivo 48 h and 10 d after infection, respectively; (b) compared with the control gliomas treated with rHSV or Endostar, the subcutaneous gliomas derived from the GSCs showed a significant reduction in microvessel density after VAE treatment; (c) compared with the control, a significant improvement was observed in the length of the survival of mice with intracranial and subcutaneous gliomas treated with VAE; (d) MRI analysis showed that the tumor volumes of the intracranial gliomas generated by GSCs remarkably decreased after 10 d of VAE treatment compared with the controls. In conclusion, VAE demonstrated oncolytic therapeutic efficacy in animal models of human GSCs and expressed an endostatin-angiostatin fusion gene, which enhanced antitumor efficacy most likely by restricting tumor microvasculature development.
Assuntos
Angiostatinas/biossíntese , Neoplasias Encefálicas/terapia , Endostatinas/biossíntese , Glioblastoma/terapia , Células-Tronco Neoplásicas/fisiologia , Simplexvirus/genética , Angiostatinas/genética , Animais , Neoplasias Encefálicas/irrigação sanguínea , Neoplasias Encefálicas/patologia , Endostatinas/genética , Terapia Genética , Glioblastoma/irrigação sanguínea , Glioblastoma/patologia , Humanos , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , Células-Tronco Neoplásicas/transplante , Neovascularização Patológica/terapia , Vírus Oncolíticos/genética , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Carga Tumoral , Células Tumorais CultivadasRESUMO
Chronic urticaria (CU) is a common disease characterized by recurrent itchy wheals and/or angioedema for more than 6 weeks. Increased levels of the pro-angiogenic mediator vascular endothelial growth factor (VEGF) have been described in skin disorders, such as chronic urticaria (CU), psoriasis and atopic dermatitis. Up to now, no data on the role of VEGF endogenous inhibitors Endostatin (ES) and Thrombospondin-1 (TSP-1) in CU are available. The aim of our study is to investigate the potential involvement of ES and TSP-1 in patients with chronic spontaneous urticaria (CSU). The levels of ES and TSP-1 were measured in the sera of 106 adult patients with CSU and 98 healthy subjects by enzyme immunoassays. The serum levels of the anti-angiogenic mediators ES and TSP-1 resulted significantly higher in CSU than in control subjects. Analysis of these mediators in CSU sub-groups, defined by the results of the autologous serum skin test (ASST), identified a significant increase of ES and TSP-1 in both ASST-positive and ASST-negative sub-groups as compared to the controls. Levels of ES and TSP-1 do not parallel the disease severity in CSU. Our study suggests that the extracellular matrix (ECM) fragments ES and TSP-1 with anti-angiogenic activity play a potential role in the pathogenesis of CSU but do not parallel disease activity.
Assuntos
Endostatinas/biossíntese , Neovascularização Patológica/prevenção & controle , Trombospondina 1/biossíntese , Urticária/sangue , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Doença Crônica , Progressão da Doença , Endostatinas/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neovascularização Patológica/etiologia , Testes Cutâneos , Trombospondina 1/sangue , Regulação para Cima , Adulto JovemRESUMO
BACKGROUND: Gene-virus targeted therapy is a promising new method of treating pancreatic cancer. To increase the efficacy and decrease the side-effect, we constructed a conditionally replicative adenovirus (CRAd) expressing human endostatin, with a human Telomoerase Reverse Transcriptase (hTERT) promoter for the regulation of the early stage of adenovirus expression of gene E1a and a Hypoxia Response Element (HRE) promoter to regulate the gene E1b. METHODS: A gene recombination technique was adopted to construct and generate the adenovirus AdTPHre-hEndo. Pancreatic cancer cells were studied both in vitro and in vivo. Western blotting was adopted to observe the expressions of protein E1A and E1B; duplication assay was applied to observe the selective duplication capability of recombinant cells. MTT assay was applied to measure the lethal effects of virus on pancreatic cancer cells, and ELISA was adopted to detect the human endostatin gene expression. A pancreatic cancer transplantation tumor model of nude mice was constructed to observe the antitumor effects of the virus. RESULTS: Double-regulated duplicative adenovirus AdTPHre-hEndo genes were successfully constructed. Duplication and lethal assays proved that AdTPHre-hEndo could replicate specifically in pancreatic cancer cells and kill them. The endostatin expression in a cultured supernatant from tumor cells was significantly higher than that obtained from non-duplicative adenovirus vectors carrying that gene. The animal experiment demonstrated that AdTPHre-hEndo has a high capability to limit pancreatic cancer growth. CONCLUSIONS: AdTPHre-hEndo has a special ability to duplicate and kill pancreatic cancer cells in in vitro and in vivo experiments, thus providing a new gene-virus-based treatment system for pancreatic cancer.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Neoplasias Pancreáticas/terapia , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/biossíntese , Proteínas E1B de Adenovirus/biossíntese , Animais , Linhagem Celular Tumoral , Endostatinas/biossíntese , Vetores Genéticos , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Replicação Viral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Previously, it was reported that the cotransfection of angiostatin K1-3, endostatin, and saxatilin genes using cationic liposomes significantly inhibited tumor progression. IL-12 is a well-known immune modulator that promotes Th1-type antitumor immune responses and also induces antiangiogenic effects. In this study, we have examined the antitumoral function of the IL-12 gene cotransfected with antiangiogenic genes for angiostatin K1-3, endostatin, and saxatilin by O,O'-dimyristyl-N-lysyl glutamate (DMKE) cationic liposomes in a mouse tumor model. According to our results, the administration of the IL-12 gene or the genes for angiostatin K1-3, endostatin, and saxatilin exhibited effective inhibition of B16BL6 melanoma growth in mice. In particular, intravenous administration of the IL-12 gene along with intratumoral administration of the three antiangiogenic genes synergistically inhibited the B16BL6 tumor growth. These results suggest that systemically expressed IL-12 enhances antitumoral efficacy of locally expressed antiangiogenic proteins.
Assuntos
Angiostatinas/genética , Desintegrinas/genética , Endostatinas/genética , Interleucina-12/genética , Melanoma Experimental/terapia , Angiostatinas/biossíntese , Animais , Processos de Crescimento Celular/genética , DNA/administração & dosagem , DNA/química , DNA/genética , Dipeptídeos/administração & dosagem , Dipeptídeos/química , Desintegrinas/biossíntese , Endostatinas/biossíntese , Feminino , Expressão Gênica , Terapia Genética/métodos , Interleucina-12/biossíntese , Lipossomos/administração & dosagem , Lipossomos/química , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/genética , Melanoma Experimental/patologia , Camundongos , Camundongos Endogâmicos C57BL , Plasmídeos/administração & dosagem , Plasmídeos/química , Plasmídeos/genética , Transfecção/métodosRESUMO
Inhibition of angiogenesis has become a particular interest for treatment of solid tumors. Endostatin, a C-terminal fragment of collagen XVIII, has been reported to exhibit potent inhibitory effect on endothelial cells proliferation, migration and tube formation. In this research, the cDNA library of endostatin was synthesized from mouse liver and inserted into the SacI and SalI enzyme-cutting sites of pUC18 cloning vector. The recombinant vector was transferred into Escherichia coli DH5a and the recombinant clone was selected on LB agar plate plus ampicillin. PCR analysis and DNA sequencing proved the presence of intact endostatin gene in pUC18. The endostatin gene subcloned into pET32a expression vector and the competent bacterial cells of E. coli BL21 were transformed by the vector harboring endostatin gene. In the optimum conditions, expression plasmid was induced with IPTG and recombinant soluble endostatin as a fusion with thioredoxin was purified with Ni-NTA (Ni(2+)-nitrilotriacetate) resin. The results showed that soluble recombinant endostatin as a fusion protein with thioredoxin is a homogenous polypeptide that inhibits angiogenesis (capillary tube formation) in human umbilical vein endothelial cells by 200 ng/ml.
Assuntos
Inibidores da Angiogênese/farmacologia , Endostatinas/farmacologia , Escherichia coli , Neovascularização Patológica/prevenção & controle , Proteínas Recombinantes/farmacologia , Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/isolamento & purificação , Animais , Capilares/efeitos dos fármacos , Capilares/patologia , Células Cultivadas , Clonagem Molecular , Ensaios de Seleção de Medicamentos Antitumorais , Endostatinas/biossíntese , Endostatinas/isolamento & purificação , Fermentação , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Camundongos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificaçãoRESUMO
Pulmonary arteriovenous malformations (PAVMs) are a common source of morbidity after bidirectional superior cavopulmonary anastomosis (Glenn). The diversion of hepatic venous effluent away from the pulmonary circulation after Glenn appears to play a significant role in the pathogenesis of PAVMs. Although the liver is known to produce factors that regulate vascular development, specific hepatic inhibitors of angiogenesis have not been described in the post-Glenn population. Endostatin, produced from its precursor collagen XVIII, is a potent inhibitor of angiogenesis produced by the liver. This study aimed to investigate the hypothesis that endostatin levels decrease in patients after Glenn. Levels of endostatin and its precursor, long-type collagen XVIII, were determined by enzyme-linked immunoassay and immunoprecipitation, respectively, for serum samples from 38 patients undergoing Glenn, total cavopulmonary anastomosis (Fontan), or biventricular repair of cardiac defects. Samples were obtained before surgery and 24 h afterward. In patients undergoing a bidirectional Glenn procedure, endostatin levels decreased after surgery (n = 17; 4.42 vs 3.34 ng/ml; p < 0.001), and long type-collagen XVIII levels increased by 200 % (n = 10; p = 0.0001). However, endostatin levels did not change after surgery in patients undergoing Fontan (n = 13) or biventricular repair (n = 8). In patients undergoing Fontan, long-type collagen XVIII increased by 18 % (p < 0.01), whereas in control subjects, the levels were unchanged. These data suggest that the diversion of hepatic blood flow away from the pulmonary circulation in patients after the Glenn procedure inhibits endostatin production from collagen XVIII, resulting in decreased circulating serum endostatin levels. A decrease in endostatin may promote angiogenesis. The mechanism whereby the pulmonary circulation processes endostatin and its potential role in the pathogenesis of PAVMs warrant further study.
Assuntos
Fístula Arteriovenosa/sangue , Endostatinas/biossíntese , Técnica de Fontan/efeitos adversos , Derivação Cardíaca Direita/efeitos adversos , Cardiopatias Congênitas/cirurgia , Neovascularização Patológica/sangue , Fístula Arteriovenosa/epidemiologia , Fístula Arteriovenosa/etiologia , Biomarcadores/sangue , Western Blotting , Pré-Escolar , Colágeno Tipo XVIII/sangue , Endostatinas/sangue , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica de Fontan/métodos , Técnica de Fontan/mortalidade , Derivação Cardíaca Direita/mortalidade , Cardiopatias Congênitas/mortalidade , Humanos , Imunoprecipitação , Lactente , Masculino , Morbidade/tendências , Neovascularização Patológica/epidemiologia , Neovascularização Patológica/etiologia , Complicações Pós-Operatórias , Artéria Pulmonar/anormalidades , Veias Pulmonares/anormalidades , Taxa de Sobrevida/tendências , Estados Unidos/epidemiologiaRESUMO
BACKGROUND: Myocardial microvascular dysfunction has been implicated in the pathogenesis of myocardial infarction (MI). We tested the hypothesis that patients with MI have lower microvasculature density in myocardium remote from the site of infarction than patients with similar extent of coronary artery disease (CAD) without MI and examined the relationship between myocardial capillary length density and plasma levels of angiogenesis-related biomarkers. METHODS: We analyzed biopsies from non-ischemic left ventricular (LV) myocardium and measured plasma levels of angiogenesis-related biomarkers in patients undergoing coronary artery bypass graft surgery, 57 without previous MI (no-MI) and 27 with recent non-ST-segment-elevation MI (NSTEMI). Comparison was made with biopsies from 31 aortic stenosis (AS) patients and 6 patients with "normal" LV without CAD. RESULTS: Myocardial microvascular density of NSTEMI patients was approximately half the density of no-MI patients, and similar to AS patients. Whereas the reduced microvascular density of AS patients was accounted for by their cardiomyocyte hypertrophy, this was not the case for NSTEMI patients, who had higher diffusion radius/cardiomyocyte width ratio than no-MI, "normal" LV, and AS patients. NSTEMI patients had lower plasma levels of carboxymethyl lysine and low molecular weight fluorophores, higher vascular endothelial growth factor (VEGF) receptor-1/VEGF-A ratio, and higher endostatin and hepatocyte growth factor levels than no-MI patients. CONCLUSIONS: Recent MI was associated with reduced microvasculature density in myocardium remote from the site of infarction and alteration in plasma levels of angiogenesis-related biomarkers.
Assuntos
Circulação Coronária/fisiologia , Microvasos/fisiologia , Infarto do Miocárdio/patologia , Miocárdio/patologia , Adulto , Idoso , Endostatinas/biossíntese , Endostatinas/sangue , Feminino , Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/sangue , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/sangue , Infarto do Miocárdio/fisiopatologia , Miocárdio/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Fator A de Crescimento do Endotélio Vascular/sangue , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/biossíntese , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/sangueRESUMO
The meniscus is a fibrocartilaginous tissue that plays an important role in controlling complex biomechanics of the knee. A perimeniscal capillary plexus supplies the outer meniscus, whereas the inner meniscus is composed of avascular tissue. Anti-angiogenic molecules, such as chondromodulin-I (ChM-I) and endostatin, have pivotal roles in preserving the avascularity of cartilage. However, the anti-angiogenic role of ChM-I is unclear in the meniscus. We hypothesized that the inner meniscus might maintain its avascular feature by expressing ChM-I. Immunohistochemical analyses revealed that ChM-I was mainly detected in the inner and superficial zones of the meniscus. On the other hand, endostatin distribution was similar between the inner and outer meniscus. In Western blot, ChM-I was detected only in the inner meniscus, whereas endostatin was equally observed in both inner and outer menisci. In addition, ChM-I concentration of the inner meniscus-derived conditioned medium was higher than that of the outer meniscus-derived medium. ChM-I removal from the inner meniscus-derived medium and functional blocking of ChM-I significantly increased endothelial cell proliferation. In this study, we demonstrated that the inner meniscus contained larger amounts of ChM-I, and that the inner meniscus-derived ChM-I inhibited endothelial cell proliferation. Our results suggest that ChM-I may be a key anti-angiogenic factor for maintaining the avascularity of the inner meniscus.
Assuntos
Proliferação de Células/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Proteínas de Membrana/fisiologia , Meniscos Tibiais/metabolismo , Idoso , Artroplastia do Joelho , Células Cultivadas , Endostatinas/biossíntese , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Proteínas de Membrana/biossínteseRESUMO
OBJECTIVE: Endostatin gene therapy for endometriosis was studied in an experimental autotransplantation model in rats. METHODS: Endometriotic lesions were transfected by intralesional injection of the plasmid lipofectamine-endostatin-pBud (group 1), lipofectamine-pBud (empty vector; group 2) or phosphate-buffered saline (group 3). Endostatin mRNA and protein levels in lesions were evaluated by quantitative real-time reverse transcription-polymerase chain reaction and Western blot analysis. Endostatin and vascular endothelial growth factor (VEGF) protein levels in serum, and microvessel density (MVD) and matrix metalloproteinase (MMP)-2 protein levels in endometriotic lesions, were also determined. RESULTS: Lipofectamine-endostatin-pBud injection increased endostatin mRNA and protein levels in lesions. Lesions were significantly smaller, and serum VEGF levels significantly lower, in group 1 versus controls. Serum VEGF was significantly and negatively correlated with serum endostatin. In group 1, MMP-2 levels and MVD were significantly lower versus controls. MMP-2 level was negatively correlated with endostatin. CONCLUSIONS: Gene therapy with endostatin appears to be an effective treatment for endometriosis. Restoration of endostatin gene expression by gene transfer in vivo might be a potential gene therapy approach for human endometriosis.
Assuntos
Inibidores da Angiogênese/genética , Endometriose/terapia , Endostatinas/genética , Terapia Genética , Inibidores da Angiogênese/biossíntese , Inibidores da Angiogênese/sangue , Animais , Endometriose/sangue , Endométrio/irrigação sanguínea , Endométrio/metabolismo , Endométrio/patologia , Endostatinas/biossíntese , Endostatinas/sangue , Feminino , Expressão Gênica , Injeções Intralesionais , Microvasos/patologia , Plasmídeos/administração & dosagem , Plasmídeos/genética , Ratos , Ratos Endogâmicos Lew , Transfecção , Fator A de Crescimento do Endotélio Vascular/sangueRESUMO
RetinoStat(®) is an equine infectious anemia virus-based lentiviral gene therapy vector that expresses the angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the wet form of age-related macular degeneration. We initiated 6-month safety and biodistribution studies in two species; rhesus macaques and Dutch belted rabbits. After subretinal administration of RetinoStat the level of human endostatin and angiostatin proteins in the vitreous of treated rabbit eyes peaked at â¼1 month after dosing and remained elevated for the duration of the study. Regular ocular examinations revealed a mild to moderate transient ocular inflammation that resolved within 1 month of dosing in both species. There were no significant long-term changes in the electroretinograms or intraocular pressure measurements in either rabbits or macaques postdosing compared with the baseline reading in RetinoStat-treated eyes. Histological evaluation did not reveal any structural changes in the eye although there was an infiltration of mononuclear cells in the vitreous, retina, and choroid. No antibodies to any of the RetinoStat vector components or the transgenes could be detected in the serum from either species, and biodistribution analysis demonstrated that the RetinoStat vector was maintained within the ocular compartment. In summary, these studies found RetinoStat to be well tolerated, localized, and capable of persistent expression after subretinal delivery.
Assuntos
Terapia Genética/métodos , Vetores Genéticos , Vírus da Anemia Infecciosa Equina , Degeneração Macular/metabolismo , Degeneração Macular/terapia , Corpo Vítreo/metabolismo , Angiostatinas/biossíntese , Angiostatinas/genética , Animais , Endostatinas/biossíntese , Endostatinas/genética , Humanos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Macaca mulatta , Degeneração Macular/patologia , Coelhos , Fatores de Tempo , Corpo Vítreo/patologia , Corpo Vítreo/virologiaRESUMO
Hydrogen sulfide (H(2)S) has recently been identified as a regulator of various physiological events, including vasodilation, angiogenesis, antiapoptotic, and cellular signaling. Endogenously, H(2)S is produced as a metabolite of homocysteine (Hcy) by cystathionine ß-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST). Although Hcy is recognized as vascular risk factor at an elevated level [hyperhomocysteinemia (HHcy)] and contributes to vascular injury leading to renovascular dysfunction, the exact mechanism is unclear. The goal of the current study was to investigate whether conversion of Hcy to H(2)S improves renovascular function. Ex vivo renal artery culture with CBS, CSE, and 3MST triple gene therapy generated more H(2)S in the presence of Hcy, and these arteries were more responsive to endothelial-dependent vasodilation compared with nontransfected arteries treated with high Hcy. Cross section of triple gene-delivered renal arteries immunostaining suggested increased expression of CD31 and VEGF and diminished expression of the antiangiogenic factor endostatin. In vitro endothelial cell culture demonstrated increased mitophagy during high levels of Hcy and was mitigated by triple gene delivery. Also, dephosphorylated Akt and phosphorylated FoxO3 in HHcy were reversed by H(2)S or triple gene delivery. Upregulated matrix metalloproteinases-13 and downregulated tissue inhibitor of metalloproteinase-1 in HHcy were normalized by overexpression of triple genes. Together, these results suggest that H(2)S plays a key role in renovasculopathy during HHcy and is mediated through Akt/FoxO3 pathways. We conclude that conversion of Hcy to H(2)S by CBS, CSE, or 3MST triple gene therapy improves renovascular function in HHcy.
Assuntos
Cistationina beta-Sintase/genética , Cistationina gama-Liase/genética , Terapia Genética , Sulfeto de Hidrogênio/metabolismo , Hiper-Homocisteinemia/terapia , Sulfurtransferases/genética , Animais , Células Cultivadas , Cistationina beta-Sintase/metabolismo , Cistationina gama-Liase/metabolismo , Endostatinas/biossíntese , Proteína Forkhead Box O3 , Fatores de Transcrição Forkhead/metabolismo , Homocisteína/metabolismo , Hiper-Homocisteinemia/genética , Hiper-Homocisteinemia/metabolismo , Hipertensão Renovascular/genética , Hipertensão Renovascular/terapia , Metaloproteinase 13 da Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Técnicas de Cultura de Órgãos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Artéria Renal/metabolismo , Sulfurtransferases/metabolismo , Inibidor Tecidual de Metaloproteinase-1/metabolismo , Fator A de Crescimento do Endotélio Vascular/biossíntese , Lesões do Sistema VascularRESUMO
OBJECTIVE: To determine the effects of carbon dioxide (CO(2)) and nitrogen (N(2)) pneumoperitoneums on endometriosis (EMs) lesions. METHODS: Female Wistar rats were randomized into the following 3 groups: CO(2) (N = 20), N(2) (N = 22) and air pneumoperitoneums (N = 9). After 5 weeks of establishment models, do the pneumoperitoneums. Then measure the size of EMs lesions and the related factors of serum and tissue after 1, 2, and 4 weeks of pneumoperitoneums. RESULTS: (1) One week after the pneumoperitoneum was established, the EMs lesions in the CO(2) group were largest in volume, whereas at 4 weeks the EMs lesions in the CO(2) group were smaller than the N(2) group. (2) The level of ICAM-1 and TIMP-2 of serum in CO(2) and N(2) group after 2 weeks of pneumoperitoneum were higher than air group. (3) The expression of CD44v6, ICAM-1, MMP-2 and VEGF of tissue in CO(2) and N(2) group after 1, 2 and 4 weeks of pneumoperitoneum were lower than air group, TIMP-2 and ENS were higher than air group. CONCLUSION: After a CO(2) pneumoperitoneum, EMs lesions were reduced in volume, suggesting an inhibitory effect on EMs lesions.
Assuntos
Dióxido de Carbono/uso terapêutico , Endometriose/terapia , Nitrogênio/uso terapêutico , Pneumoperitônio Artificial/métodos , Animais , Endostatinas/biossíntese , Feminino , Receptores de Hialuronatos/biossíntese , Molécula 1 de Adesão Intercelular/sangue , Metaloproteinase 2 da Matriz/biossíntese , Ratos , Ratos Wistar , Inibidor Tecidual de Metaloproteinase-2/sangue , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
A recombinant adenovirus encoding human endostatin gene, E10A, has finished phase II trials for head and neck cancer. However, the rigid storage temperature (-80°C) and the toxicity of glycerol in the E10A liquid preparation limited its clinical application. In this study, lyophilization was applied to develop a stable E10A lyophilized powder without glycerol that is able to maintain biological activity at 4°C and suitable for intravenous administration. The E10A lyophilized formulations composed of nontoxic and already clinically used excipients were characterized in terms of the pH change during freezing, the eutectic melting temperature (T(eu)) and the collapse temperature (T(c)). Freeze thawing tests were carried out to examine the protective effect of various excipients during freezing. Mannitol and its combinations with sucrose or inulin showed effective protection of E10A. The E10A lyophilized powders were analyzed by particle size measurement, residual humidity quantification, infectivity assay and gene expression level. An optimized formulation (formulation I1) yielded a good recovery of 76% of the starting infectivity after lyophilization and 89% of the original infectivity after storage at 4°C for 180 days. Also the gene expression capability of E10A in formulation I1 was maintained after lyophilization. In addition, it was found that the matrix of amorphous excipients, mannitol combinations with sucrose or inulin, was indispensible in protecting E10A against the stress of freezing and dehydration. Hereby, the E10A lyophilized powder with eliminated glycerol toxicity and improved stability could enhance the applicability of E10A for cancer gene therapy through intravenous administration.
Assuntos
Adenoviridae/genética , Endostatinas/genética , Terapia Genética/métodos , Vetores Genéticos , Linhagem Celular , Química Farmacêutica , Cristalização , Estabilidade de Medicamentos , Endostatinas/biossíntese , Excipientes , Liofilização , Congelamento , Expressão Gênica , Humanos , Umidade , Concentração de Íons de Hidrogênio , Injeções Intravenosas , Lasers , Tamanho da Partícula , Pós , Espalhamento de Radiação , Temperatura , Difração de Raios XRESUMO
We have recently demonstrated that a 4-in-1 gene therapy strategy that contains two anti-angiogenic genes [endostatin and pigment epithelium-derived factor] and two cytokine genes [granulocyte macrophage colony-stimulating factor and interleukin 12] has a considerable antitumor effect on large tumors in a woodchuck hepatoma model. The current study further investigates the underlying mechanisms for the antitumor effect observed by using small rodent models. We found that immunotherapy alone increased immunosuppressive cells in large tumors over time, whereas the anti-angiogenic therapy contained in the 4-in-1 strategy alleviated immunosuppression and made tumors vulnerable to immunotherapy, thus resulting in a synergistic antitumor effect.
Assuntos
Endostatinas/genética , Endostatinas/imunologia , Proteínas do Olho/genética , Proteínas do Olho/imunologia , Terapia Genética/métodos , Imunoterapia/métodos , Neoplasias Hepáticas Experimentais/terapia , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/imunologia , Serpinas/genética , Serpinas/imunologia , Adenoviridae/genética , Animais , Apoptose/genética , Apoptose/imunologia , Linhagem Celular Tumoral , Terapia Combinada , Endostatinas/biossíntese , Proteínas do Olho/biossíntese , Humanos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/imunologia , Linfócitos do Interstício Tumoral/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Patológica/genética , Neovascularização Patológica/imunologia , Neovascularização Patológica/terapia , Fatores de Crescimento Neural/biossíntese , Serpinas/biossíntese , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologiaRESUMO
Most cancer chemotherapeutic agents are administered at the maximum-tolerated dose (MTD) in short cycles with treatment breaks. However, MTD-based chemotherapies are often associated with significant toxicity and treatment breaks allow the opportunity for tumor regrowth and acquisition of chemoresistance. To minimize these drawbacks, a metronomic strategy, in which chemotherapeutics are administered at doses significantly below the MTD without treatment breaks, has been suggested by many investigators. The antitumor effect of metronomic chemotherapy may be partially due to inhibition of tumor angiogenesis, and it could be enhanced by a combination therapy, including antiangiogenic agents. In this study, we evaluated the synergistic effect of E10A, an adenovirus carrying the endostatin gene, the most potent inhibitors of tumor angiogenesis, in combination with weekly low-dose cisplatin in a xenograft mouse model for head and neck squamous-cell carcinoma. The E10A induced mRNA and protein expressions of endostatin in H891 cells in vitro. E10A significantly enhanced the in vivo tumor growth inhibitory effect of cisplatin. Immunohistochemical analysis with a TUNEL (terminal deoxynucleotidyl transferase-mediated nick-end labeling) assay and anti-CD31 antibodies revealed that the combination of E10A and cisplatin induced high levels of cell apoptosis and inhibited tumor angiogenesis. Importantly, E10A increased the platinum concentrations in tumors to fivefold higher than that induced by cisplatin alone.