RESUMO
BACKGROUND: Tumor vascular mimicry is an emerging issue that affects patient survival while having no treatment at the current moment. Despite several factors implicated in vascular mimicry, little is known about stromal factors that modulate tumor microenvironment and shape malignant transformation. CD248, a type-I transmembrane protein dominantly expressed in stromal cells, mediates the interaction between cells and extracellular matrix proteins. CD248 protein expression is associated with the metastatic melanoma phenotype and promotes tumor progression in the stromal cells. This study aimed to explore the cell-autonomous effects of CD248 in melanoma vascular mimicry to aid cancer therapy development. METHODS: Loss-of-function approaches in B16F10 melanoma cells were used to study the cell-autonomous effects of CD248 on cell adhesion, migration, proliferation, and vascular mimicry. A solid-phase binding assay was performed to identify the interaction between CD248 and fibronectin. Horizontal and vertical cell migration assays were performed to analyze cell migration activity, and cell-patterned network formation on Matrigel was used to evaluate vascular mimicry activity. Recombinant CD248 (rCD248) proteins were generated, and whether rCD248 interfered with melanoma CD248 functions was evaluated in vitro. An experimental lung metastasis mouse model was used to investigate the effect of rCD248 treatment in vivo. RESULTS: CD248 protein expression in melanoma cells was increased by a fibroblast-conditioned medium. Knockdown of CD248 expression significantly decreased cell adhesion to fibronectin, cell migration, and vascular mimicry in melanoma cells. The lectin domain of CD248 was directly involved in the interaction between CD248 and fibronectin. Furthermore, rCD248 proteins containing its lectin domain inhibited cell adhesion to fibronectin and slowed down cell migration and vascular mimicry. Treatment with rCD248 protein could reduce pulmonary tumor burden, accompanied by a reduction in vascular mimicry in mice with melanoma lung metastasis. CONCLUSION: CD248 expression in melanoma cells promotes malignant transformation by increasing the activity of cell adhesion, migration, and vascular mimicry, whereas rCD248 protein functions as a molecular decoy interfering with tumor-promoting effects of CD248 in melanoma cells.
Assuntos
Neoplasias Pulmonares , Melanoma , Camundongos , Animais , Fibronectinas , Melanoma/genética , Adesão Celular , Neoplasias Pulmonares/genética , Lectinas/farmacologia , Microambiente Tumoral , Antígenos de Neoplasias/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/farmacologiaRESUMO
OBJECTIVE: Thrombomodulin (TM), a glycoprotein constitutively expressed in the endothelium, is well known for its anticoagulant and anti-inflammatory properties. Paradoxically, we recently found that monocytic membrane-bound TM (ie, endogenous TM expression in monocytes) triggers lipopolysaccharide- and gram-negative bacteria-induced inflammatory responses. However, the significance of membrane-bound TM in chronic sterile vascular inflammation and the development of abdominal aortic aneurysm (AAA) remains undetermined. APPROACH AND RESULTS: Implicating a potential role for membrane-bound TM in AAA, we found that TM signals were predominantly localized to macrophages and vascular smooth muscle cells in human aneurysm specimens. Characterization of the CaCl2-induced AAA in mice revealed that during aneurysm development, TM expression was mainly localized in infiltrating macrophages and vascular smooth muscle cells. To investigate the function of membrane-bound TM in vivo, transgenic mice with myeloid- (LysMcre/TM(flox/flox)) and vascular smooth muscle cell-specific (SM22-cre(tg)/TM(flox/flox)) TM ablation and their respective wild-type controls (TM(flox/flox) and SM22-cre(tg)/TM(+/+)) were generated. In the mouse CaCl2-induced AAA model, deficiency of myeloid TM, but not vascular smooth muscle cell TM, inhibited macrophage accumulation, attenuated proinflammatory cytokine and matrix metalloproteinase-9 production, and finally mitigated elastin destruction and aortic dilatation. In vitro TM-deficient monocytes/macrophages, versus TM wild-type counterparts, exhibited attenuation of proinflammatory mediator expression, adhesion to endothelial cells, and generation of reactive oxygen species. Consistently, myeloid TM-deficient hyperlipidemic mice (ApoE(-/-)/LysMcre/TM(flox/flox)) were resistant to AAA formation induced by angiotensin II infusion, along with reduced macrophage infiltration, suppressed matrix metalloproteinase activities, and diminished oxidative stress. CONCLUSIONS: Membrane-bound TM in macrophages plays an essential role in the development of AAA by enhancing proinflammatory mediator elaboration, macrophage recruitment, and oxidative stress.
Assuntos
Aorta Abdominal/metabolismo , Aneurisma da Aorta Abdominal/metabolismo , Aortite/metabolismo , Membrana Celular/metabolismo , Macrófagos Peritoneais/metabolismo , Trombomodulina/metabolismo , Angiotensina II , Animais , Aorta Abdominal/imunologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/imunologia , Aortite/induzido quimicamente , Aortite/genética , Aortite/imunologia , Cloreto de Cálcio , Membrana Celular/imunologia , Células Cultivadas , Quimiotaxia , Modelos Animais de Doenças , Elastina/metabolismo , Células Endoteliais da Veia Umbilical Humana/imunologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Macrófagos Peritoneais/imunologia , Metaloproteinase 9 da Matriz/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/imunologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/imunologia , Miócitos de Músculo Liso/metabolismo , Estresse Oxidativo , Interferência de RNA , Estudos Retrospectivos , Transdução de Sinais , Trombomodulina/deficiência , Trombomodulina/genética , Fatores de Tempo , TransfecçãoRESUMO
Lewis Y Ag (LeY) is a cell-surface tetrasaccharide that participates in angiogenesis. Recently, we demonstrated that LeY is a specific ligand of the recombinant lectin-like domain of thrombomodulin (TM). However, the biologic function of interaction between LeY and TM in endothelial cells has never been investigated. Therefore, the role of LeY in tube formation and the role of the recombinant lectin-like domain of TM-TM domain 1 (rTMD1)-in antiangiogenesis were investigated. The recombinant TM ectodomain exhibited lower angiogenic activity than did the recombinant TM domains 2 and 3. rTMD1 interacted with soluble LeY and membrane-bound LeY and inhibited soluble LeY-mediated chemotaxis of endothelial cells. LeY was highly expressed on membrane ruffles and protrusions during tube formation on Matrigel. Blockade of LeY with rTMD1 or Ab against LeY inhibited endothelial tube formation in vitro. Epidermal growth factor (EGF) receptor in HUVECs was LeY modified. rTMD1 inhibited EGF receptor signaling, chemotaxis, and tube formation in vitro, and EGF-mediated angiogenesis and tumor angiogenesis in vivo. We concluded that LeY is involved in vascular endothelial tube formation and rTMD1 inhibits angiogenesis via interaction with LeY. Administration of rTMD1 or recombinant adeno-associated virus vector carrying TMD1 could be a promising antiangiogenesis strategy.
Assuntos
Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Proteínas Recombinantes/farmacologia , Trombomodulina/química , Animais , Carcinoma Pulmonar de Lewis/tratamento farmacológico , Carcinoma Pulmonar de Lewis/patologia , Células Cultivadas , Regulação para Baixo/efeitos dos fármacos , Feminino , Células HEK293 , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Células Endoteliais da Veia Umbilical Humana/transplante , Humanos , Lectinas/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Fisiológica/fisiologia , Ligação Proteica , Estrutura Terciária de Proteína/fisiologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapêutico , Trombomodulina/metabolismo , Trombomodulina/uso terapêutico , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Urokinase-type plasminogen activator (uPA) activates plasminogen (Plg) through a major pericellular proteolytic system involved in cell migration and angiogenesis; however, the Plg receptor that participates in uPA-mediated Plg activation has not yet been identified. In this study, we demonstrated that thrombomodulin (TM), a type I transmembrane glycoprotein, is a novel Plg receptor that plays a role in pericellular proteolysis and cell migration. Plg activation at the cell surface and the extent of its cell migration- and invasion-promoting effect are cellular TM expression dependent. Direct binding of Plg and the recombinant TM extracellular domain, with a KD of 0.1-0.3 µM, was determined through surface plasmon resonance analysis. Colocalization of TM, Plg, and the uPA receptor within plasma membrane lipid rafts, at the leading edge of migrating endothelial cells, was demonstrated and was also shown to overlap with areas of major pericellular proteolysis. Moreover, the roles of TM and Plg in neoangiogenesis were demonstrated in vivo through the skin wound-healing model. In conclusion, we propose that TM is a novel Plg receptor that regulates uPA/uPA receptor-mediated Plg activation and pericellular proteolysis within lipid rafts at the leading edge of migrating cells during angiogenesis.
Assuntos
Neovascularização Fisiológica , Plasminogênio/metabolismo , Trombomodulina/metabolismo , Animais , Células CHO , Movimento Celular , Cricetinae , Cricetulus , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Microdomínios da Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Fator de Crescimento Placentário , Proteínas da Gravidez/genética , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteólise , Pele/irrigação sanguínea , Trombomodulina/química , Trombomodulina/genética , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , CicatrizaçãoRESUMO
Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR) signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2), an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis.
Assuntos
Anoikis , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Serina Proteases/metabolismo , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Serina EndopeptidasesRESUMO
Tumor endothelial marker 1 (TEM1), an activated mesenchymal cell marker, is implicated in tissue remodeling and repair. Herein, we investigated the role and therapeutic implications of TEM1 in abdominal aortic aneurysm (AAA), a potentially life-threatening aortic disease characterized by vascular inflammation and matrix turnover. Characterization of human AAA revealed increased TEM1 expression derived mainly from medial vascular smooth muscle cells (VSMCs) and adventitial fibroblasts. Bioinformatics analysis demonstrated the association between TEM1-expressing VSMCs and fibroblasts and collagen gene expression. Consistently, collagen content and TEM1 expressed by VSMCs and fibroblasts were increased during CaCl2-induced AAA formation in mice. TEM1 silencing in VSMCs and fibroblasts inhibited transforming growth factor-ß1-induced phenotypic change, SMAD2 phosphorylation, and COL1A1 gene expression. Also, Tem1 deficiency reduced collagen synthesis and exacerbated CaCl2-induced AAA formation in mice without disturbing elastin destruction and inflammatory responses. In contrast, rTEM1 promoted phenotypic change and COL1A1 gene expression through SMAD2 phosphorylation in VSMCs and fibroblasts. Treatment with rTEM1 enhanced collagen synthesis, attenuated elastin fragmentation, and inhibited CaCl2-induced and angiotensin II-infused AAA formation. In summary, TEM1 in resident stromal cells regulates collagen synthesis to counteract aortic wall failure during AAA formation. Matrix integrity restored by rTEM1 treatment may hold therapeutic potential against AAA.
Assuntos
Aneurisma da Aorta Abdominal , Animais , Humanos , Masculino , Camundongos , Aneurisma da Aorta Abdominal/metabolismo , Fibroblastos/metabolismo , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína Smad2/metabolismoRESUMO
OBJECTIVE: To investigate whether recombinant thrombomodulin containing all the extracellular domains (rTMD123) has therapeutic potential against aneurysm development. SUMMARY BACKGROUND DATA: The pathogenesis of abdominal aortic aneurysm (AAA) is characterized by chronic inflammation and proteolytic degradation of extracellular matrix. Thrombomodulin, a transmembrane glycoprotein, exerts anti-inflammatory activities such as inhibition of cytokine production and sequestration of proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that may sustain inflammation and tissue damage. METHODS: The in vivo effects of treatment and posttreatment with rTMD123 on aortic dilatation were measured using the CaCl2-induced AAA model in mice. RESULTS: Characterization of the CaCl2-induced model revealed that HMGB1 and RAGE, both localized mainly to macrophages, were persistently upregulated during a 28-day period of AAA development. In vitro, rTMD123-HMGB1 interaction prevented HMGB1 binding to macrophages, thereby prohibiting activation of HMGB1-RAGE signaling in macrophages. In vivo, short-term treatment with rTMD123 upon AAA induction suppressed the levels of proinflammatory cytokines, HMGB1, and RAGE in the aortic tissue; reduced the infiltrating macrophage number; and finally attenuated matrix metalloproteinase production, extracellular matrix destruction, and AAA formation without disturbing vascular calcification. Consistently, posttreatment with rTMD123 seven days after AAA induction alleviated vascular inflammation and retarded AAA progression. CONCLUSIONS: These data suggest that rTMD123 confers protection against AAA development. The mechanism of action may be associated with reduction of proinflammatory mediators, blockade of macrophage recruitment, and suppression of HMGB1-RAGE signaling involved in aneurysm formation and downstream macrophage activation.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Trombomodulina/uso terapêutico , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Cloreto de Cálcio/administração & dosagem , Camundongos , Camundongos Endogâmicos C57BL , Receptor para Produtos Finais de Glicação Avançada , Receptores Imunológicos/fisiologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Rationale: CD93, a C-type lectin-like transmembrane glycoprotein, can be shed in a soluble form (sCD93) upon inflammatory stimuli. sCD93 effectively enhances apoptotic cell clearance and has been proposed as an inflammatory disease biomarker. The function of sCD93 involved directly in inflammation remains to be determined. Herein, we attempted to examine the hypothesis that sCD93 might sequester proinflammatory high-mobility group box 1 protein (HMGB1), exerting anti-inflammatory properties. Methods: Different forms of soluble recombinant human CD93 (rCD93) were prepared by a mammalian protein expression system. rCD93-HMGB1 interaction was assessed using co-immunoprecipitation and solid-phase binding assays. Effects of soluble rCD93 were evaluated in HMGB1-induced macrophage and vascular smooth muscle cells (VSMC) activation and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis, CaCl2-induced and angiotensin II-infused abdominal aortic aneurysm (AAA) formation and ovariectomized-induced osteoporosis in mice. Results: Protein binding studies revealed that soluble rCD93, via the lectin-like domain (D1), can bind to HMGB1 and intercept HMGB1-receptor interaction. Soluble rCD93 containing D1 inhibited HMGB1-induced proinflammatory cytokine production and intracellular mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB activation in macrophages and VSMCs, thereby attenuating CaCl2-induced and angiotensin II-infused AAA models. During osteoclastogenesis, RANKL stimulated HMGB1 secretion that promoted RANKL-induced osteoclastogenesis in return. Soluble rCD93 containing D1 impeded RANKL-induced osteoclastogenic marker gene expression and intracellular MAPK/NF-κB signaling, thereby mitigating ovariectomized-induced osteoporosis. Conclusion: These findings demonstrate the therapeutic potential of soluble recombinant CD93 containing D1 in inflammatory diseases. Our study highlights a novel anti-inflammatory mechanism, i.e., sequestration of HMGB1, through which sCD93 prevents HMGB1-receptor interaction on effector cells and alleviates inflammation.
Assuntos
Proteína HMGB1 , Humanos , Animais , Camundongos , Proteína HMGB1/metabolismo , Lectinas , Angiotensina II , Cloreto de Cálcio , Inflamação , Mamíferos/metabolismoRESUMO
BACKGROUND: Evidence demonstrates that the thrombomodulin (TM) lectin domain (TMD1) exerts anti-inflammatory functions. Lipopolysaccharides derived from Porphyromonas gingivalis (Pg-LPS) are considered a major pathogenic factor for chronic periodontitis, promoting inflammation, osteoclastogenesis and alveolar bone resorption. Herein, we aimed to evaluate the potential therapeutic effect of recombinant TMD1 (rTMD1) in suppression of Pg-LPS-induced osteoclastogenesis and periodontal bone loss. METHODS: In vitro, the effects of Pg-LPS, tumor necrosis factor (TNF)-α and rTMD1 on osteoclast differentiation were investigated using receptor activator of nuclear factor-κB ligand (RANKL)-stimulated RAW 264.7 macrophages. In vivo, the effects of rTMD1 treatment were evaluated in a model of experimental periodontitis induced by direct injection of Pg-LPS into the vestibular gingiva. RESULTS: Administration of Pg-LPS to RANKL-stimulated RAW 264.7 macrophages resulted in upregulation of CD86 and osteoclast marker (eg, Dc-stamp and Trap) gene expression and increase of pro-inflammatory cytokine production (e.g., TNF-α) during osteoclast differentiation, and rTMD1 can attenuate these effects. Also, rTMD1 inhibited Pg-LPS-enhanced in vitro bone resorption in a dose-dependent manner. Moreover, TNF-α promoted phosphorylation of p38 and ERK during osteoclast differentiation, and the signal activation can be inhibited by rTMD1. Finally, treatment with rTMD1 hindered Pg-LPS-induced alveolar bone loss in experimental periodontitis in mice. CONCLUSION: Our study demonstrated that rTMD1 attenuates Pg-LPS-enhanced M1 macrophage polarization, osteoclastogenesis and periodontal bone resorption and thus holds therapeutic promise for periodontitis.
Assuntos
Perda do Osso Alveolar , Reabsorção Óssea , Perda do Osso Alveolar/tratamento farmacológico , Perda do Osso Alveolar/prevenção & controle , Animais , Reabsorção Óssea/tratamento farmacológico , Diferenciação Celular , Lectinas , Lipopolissacarídeos , Camundongos , Osteoclastos , Osteogênese , Porphyromonas gingivalis , Ligante RANK , TrombomodulinaRESUMO
Thrombomodulin (TM), a widely expressing glycoprotein originally identified in vascular endothelium, is an important cofactor in the protein C anticoagulant system. TM appears to exhibit anti-inflammatory ability through both protein C-dependent and -independent pathways. We presently have demonstrated that recombinant N-terminal lectinlike domain of TM (rTMD1) functions as a protective agent against sepsis caused by Gram-negative bacterial infections. rTMD1 caused agglutination of Escherichia coli and Klebsiella pneumoniae and enhanced the macrophage phagocytosis of these Gram-negative bacteria. Moreover, rTMD1 bound to the Klebsiella pneumoniae and lipopolysaccharide (LPS) by specifically interacting with Lewis Y antigen. rTMD1 inhibited LPS-induced inflammatory mediator production via interference with CD14 and LPS binding. Furthermore, rTMD1 modulated LPS-induced mitogen-activated protein kinase and nuclear factor-kappaB signaling pathway activations and inducible nitric oxide synthase expression in macrophages. Administration of rTMD1 protected the host by suppressing inflammatory responses induced by LPS and Gram-negative bacteria, and enhanced LPS and bacterial clearance in sepsis. Thus, rTMD1 can be used to defend against bacterial infection and inhibit LPS-induced inflammatory responses, suggesting that rTMD1 may be valuable in the treatment of severe inflammation in sepsis, especially in Gram-negative bacterial infections.
Assuntos
Antígenos do Grupo Sanguíneo de Lewis/metabolismo , Trombomodulina/química , Trombomodulina/metabolismo , Animais , Sítios de Ligação , Linhagem Celular , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Inflamação/tratamento farmacológico , Inflamação/prevenção & controle , Klebsiella pneumoniae/metabolismo , Klebsiella pneumoniae/patogenicidade , Ligantes , Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/toxicidade , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos , NF-kappa B/metabolismo , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Sepse/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Trombomodulina/administração & dosagemRESUMO
Tumor endothelial marker 1 (TEM1), also known as endosialin or CD248, is a type I transmembrane glycoprotein containing a C-type lectin-like domain. It is highly expressed in pericytes and fibroblasts. Dermal fibroblasts play a pivotal role during cutaneous wound healing, especially in the proliferative phase. However, the physiological function of TEM1 in wound healing is still undetermined. During the process of wound healing, the expression of both TEM1 and platelet-derived growth factor (PDGF) receptor α was highly upregulated in myofibroblasts. In vivo, fibroblast activation and collagen deposition in granulation tissues were attenuated, and wound healing was retarded in TEM1-deleted mice. In vitro, the migration, adhesion, and proliferation of NIH3T3 cells were suppressed following TEM1 knockdown by short hairpin RNA. In PDGF-BB-treated NIH3T3 cells, the downstream signal and mitogenic, and chemoattractive effects were inhibited by TEM1 knockdown. In addition, TEM1 and PDGF receptor α were colocalized in subcellular organelles in fibroblasts, and the association of TEM1 and PDGF receptor α was demonstrated by coimmunoprecipitation. In summary, these findings suggested that TEM1, in combination with PDGF receptor α, plays a critical role in wound healing by enhancing the mitogenic and chemoattractive effects of PDGF-BB and collagen deposition in myofibroblasts.
Assuntos
Antígenos CD/genética , Regulação da Expressão Gênica , Proteínas de Neoplasias/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Cicatrização/genética , Ferimentos e Lesões/patologia , Animais , Western Blotting/métodos , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Fatores de Tempo , Resultado do Tratamento , Regulação para Cima , Cicatrização/fisiologia , Ferimentos e Lesões/metabolismoRESUMO
Salviae miltiorrhizae (SM), a clinical, commonly used herb, can activate blood circulation and resolve stasis. We have investigated the effects of salvianolic acid B (Sal B), a pure compound extracted from the dried SM roots, on fibrinolytic (tissue-type plasminogen activator and plasminogen activator inhibitor, t-PA and PAI) and anticoagulant (thrombomodulin,TM) properties of cultured human umbilical vein endothelial cells (HUVECs). When HUVECs were treated with Sal B, a dose- (0.0125-0.5 mg/ml) and a time-dependent decrease in PAI activity were observed. PAI type 1 (PAI-1) antigen and PAI-1 mRNA expression significantly decreased compared to control values in the conditioned media of HUVECs pretreated with Sal B for 12 h. Moreover, TM activity reached a maximum stimulation of 1.25-fold over control levels in the pretreatment of Sal B for 12 h and t-PA and TM specific mRNA expression also increased (1.7- and 1.8-fold, respectively). In conclusion, Sal B increased the fibrinolytic and anticoagulant potential of cultured HUVECs by up-regulating the expression of t-PA and TM and by down-regulating the expression of PAI-1. These data suggest that Sal B is clinically effective because of its ability to change the gene expression profile of endothelial cells thereby preventing vascular events.
Assuntos
Benzofuranos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Células Endoteliais/fisiologia , Hemostasia/efeitos dos fármacos , Veias Umbilicais/fisiologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Fibrinolíticos/metabolismo , Humanos , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Inativadores de Plasminogênio/metabolismo , RNA Mensageiro/metabolismo , Trombomodulina/efeitos dos fármacos , Trombomodulina/genética , Trombomodulina/fisiologia , Ativador de Plasminogênio Tecidual/genética , Veias Umbilicais/citologia , Veias Umbilicais/metabolismoRESUMO
BACKGROUND AND AIMS: Thrombomodulin (TM), through its lectin-like domain (TMD1), sequesters proinflammatory high-mobility group box 1 (HMGB1) to prevent it from engaging the receptor for advanced glycation end product (RAGE) that sustains inflammation and tissue damage. Our previous study demonstrated that short-term treatment with recombinant TM containing all the extracellular domains (i.e., rTMD123) inhibits HMGB1-RAGE signaling and confers protection against CaCl2-induced AAA formation. In this study, we attempted to further optimize TM domains, as a potential therapeutic agent for AAA, using the recombinant adeno-associated virus (AAV) vector. METHODS: The therapeutic effects of recombinant TMD1 (rTMD1) and recombinant AAV vectors carrying the lectin-like domain of TM (rAAV-TMD1) were evaluated in the CaCl2-induced AAA model and angiotensin II-infused AAA model, respectively. RESULTS: In the CaCl2-induced model, treatment with rTMD1 suppressed the tissue levels of HMGB1 and RAGE, macrophage accumulation, elastin destruction and AAA formation, and the effects were comparable to a mole-equivalent dosage of rTMD123. In the angiotensin II-infused model, a single intravenous injection of rAAV-TMD1 (1011 genome copies), which resulted in a persistently high serum level of TMD1 for at least 12 weeks, effectively attenuated AAA formation with suppression of HMGB1 and RAGE levels and inhibition of proinflammatory cytokine production, macrophage accumulation, matrix metalloproteinase activities and oxidative stress in the aortic wall. CONCLUSIONS: These findings corroborate the therapeutic potential of the TM lectin-like domain in AAA. The attenuation of angiotensin II-infused AAA by one-time delivery of rAAV-TMD1 provides a proof-of-concept validation of its application as potential gene therapy for aneurysm development.
Assuntos
Aneurisma da Aorta Abdominal/prevenção & controle , Dependovirus/genética , Terapia Genética/métodos , Vetores Genéticos , Trombomodulina/genética , Angiotensina II , Animais , Aorta Abdominal/metabolismo , Aorta Abdominal/patologia , Aneurisma da Aorta Abdominal/induzido quimicamente , Aneurisma da Aorta Abdominal/genética , Aneurisma da Aorta Abdominal/metabolismo , Cloreto de Cálcio , Citocinas/metabolismo , Modelos Animais de Doenças , Elastina/metabolismo , Proteína HMGB1/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Knockout para ApoE , Estresse Oxidativo , Domínios Proteicos , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Trombomodulina/biossíntese , Trombomodulina/metabolismo , Remodelação VascularRESUMO
Toll-like receptor (TLR) family plays a key role in innate immunity and various inflammatory responses. TLR4, one of the well-characterized pattern-recognition receptors, can be activated by endogenous damage-associated molecular pattern molecules such as high mobility group box 1 (HMGB1) to sustain sterile inflammation. Evidence suggested that blockade of TLR4 signaling may confer protection against abdominal aortic aneurysm (AAA). Herein we aimed to obtain further insight into the mechanism by which TLR4 might promote aneurysm formation. Characterization of the CaCl2-induced AAA model in mice revealed that upregulation of TLR4 expression, localized predominantly to vascular smooth muscle cells (VSMCs), was followed by a late decline during a 28-day period of AAA development. In vitro, TLR4 expression was increased in VSMCs treated with HMGB1. Knockdown of TLR4 by siRNA attenuated HMGB1-enhanced production of proinflammatory cytokines, specifically interleukin-6 and monocyte chemoattractant protein-1 (MCP-1), and matrix-degrading matrix metalloproteinase (MMP)-2 from VSMCs. In vivo, two different strains of TLR4-deficient (C57BL/10ScNJ and C3H/HeJ) mice were resistant to CaCl2-induced AAA formation compared to their respective controls (C57BL/10ScSnJ and C3H/HeN). Knockout of TLR4 reduced interleukin-6 and MCP-1 levels and HMGB1 expression, attenuated macrophage accumulation, and eventually suppressed MMP production, elastin destruction and VSMC loss. Finally, human AAA exhibited higher TLR4 expression that was localized to VSMCs. These data suggest that TLR4 signaling contributes to AAA formation by promoting a proinflammatory status of VSMCs and by inducing proteinase release from VSMCs during aneurysm initiation and development.
Assuntos
Aneurisma da Aorta Abdominal/metabolismo , Receptor 4 Toll-Like/fisiologia , Animais , Aneurisma da Aorta Abdominal/induzido quimicamente , Cloreto de Cálcio , Estudos de Casos e Controles , Células Cultivadas , Citocinas/biossíntese , Proteína HMGB1/metabolismo , Humanos , Masculino , Metaloproteinases da Matriz/metabolismo , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Receptor para Produtos Finais de Glicação Avançada/metabolismo , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Regulação para CimaRESUMO
Angiostatin and other plasminogen derivatives exhibit antitumor activities directly or indirectly, have demonstrated promising anticancer effects in preclinical studies, but have mostly failed in clinical trials partly due to their short serum half-lives. Our previous studies demonstrated that recombinant human plasminogen kringle 1-5 (K1-5) has superior antitumor activity compared with angiostatin. In addition, optimization of recombinant K1-5 with three amino acid substitutions enhances its antitumor effect. The current study was thus undertaken to evaluate prolonged expression of optimized K1-5 as cancer gene therapy. The recombinant adeno-associated virus (AAV) vector was used to express a secreted form of the optimized K1-5 (AAV-sK15tm) to improve its pharmacokinetic profile, which was considered to be the hurdle in angiostatin treatment of cancer. We successfully generated high-titer recombinant AAV vectors and observed sustained transgene expression for 567 days after a single injection of virus. The treated animals did not display any visible signs of abnormalities and showed normal serum biochemistry. The therapeutic potential of this treatment modality was demonstrated by both a strong inhibition of lung metastasis in the mouse B16F10 melanoma model and significant growth retardation of Lewis lung carcinoma xenografts in C57BL/6N mice as well as human A2058 melanoma xenografts in NOD/SCID (nonobese diabetic/severe combined immunodeficient) mice. Taken together, our results suggested that AAV-sK15tm produced long-term suppressive effects on cancer growth in vivo and should warrant serious consideration for clinical development.
Assuntos
Carcinoma Pulmonar de Lewis/terapia , Dependovirus/genética , Terapia Genética , Neoplasias Pulmonares/prevenção & controle , Melanoma/terapia , Plasminogênio/genética , Animais , Apoptose , Expressão Gênica , Células HEK293 , Humanos , Neoplasias Pulmonares/secundário , Masculino , Melanoma/patologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos NOD , Camundongos SCID , Transplante de Neoplasias , Neovascularização Patológica , Estrutura Terciária de ProteínaRESUMO
AIMS: The recombinant epidermal growth factor-like domain plus the serine/threonine-rich domain of thrombomodulin (rTMD23) promotes angiogenesis and accelerates the generation of activated protein C (APC), which facilitates angiogenesis. The aim of this study was to elucidate the molecular mechanisms underlying the angiogenic activity of rTMD23. METHODS AND RESULTS: We prepared rTMD23 and its mutants that did not possess the ability to promote APC generation and investigated their angiogenic activities in vitro and in vivo. rTMD23 mutants promoted proliferation, migration, and tube formation of human umbilical vein endothelial cells in vitro and induced neovascularization in vivo; these effects were similar to those exerted by wild-type rTMD23. To investigate its interaction with rTMD23, Type I fibroblast growth factor receptor (FGFR1) was precipitated along with syndecan-4 by rTMD23-conjugated Sepharose in human umbilical vein endothelial cells and FGFR1-expressing human embryonic kidney 293 cells. Additionally, the kinetics of the interaction between rTMD23 and FGFR1 were analysed using surface plasmon resonance. rTMD23-induced FGFR1 activation and tube formation were inhibited by an FGFR1-specific tyrosine kinase inhibitor, PD173074, or by knockdown of FGFR1 using siRNA technology. We observed an improvement in rat hindlimb recovery in an ischaemic model following rTMD23 treatment, and this was associated with increased neovascularization and FGFR1 phosphorylation. CONCLUSION: rTMD23 induced angiogenesis via FGFR1, a process that is independent of the APC pathway.
Assuntos
Neovascularização Fisiológica/fisiologia , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Trombomodulina/fisiologia , Indutores da Angiogênese/química , Indutores da Angiogênese/farmacologia , Animais , Neovascularização da Córnea/etiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Membro Posterior/irrigação sanguínea , Células Endoteliais da Veia Umbilical Humana , Humanos , Isquemia/tratamento farmacológico , Isquemia/patologia , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos BALB C , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Proteína C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Sindecana-4/metabolismo , Trombomodulina/química , Trombomodulina/genéticaRESUMO
Keratinocyte-expressed thrombomodulin (TM) and the released soluble TM (sTM) have been demonstrated to promote wound healing. However, the effects of high glucose on TM expression in keratinocytes and the role of TM in diabetic ulcer remain unclear. In this study, we demonstrated that expressions of TM and Toll-like receptor 4 (TLR4) were both downregulated in high-glucose cultured human keratinocytes and in skin keratinocytes of diabetic patients. In addition, the wound-triggered upregulation of TM and sTM production was abolished in both high-glucose cultured human keratinocytes and streptozotocin-induced diabetic mouse skin. Furthermore, supplementation of recombinant sTM could increase TLR4 expression and promote cutaneous wound healing in both high-glucose cultured human keratinocytes and diabetic mice. However, in Tlr4-deleted mice, which exhibited delayed wound healing, the therapeutic benefit of recombinant sTM was abrogated. Moreover, our results showed that tumor necrosis factor-α (TNF-α) expression in keratinocytes was dose-dependently upregulated by glucose, and TNF-α treatment downregulated the expression of TM and TLR4. Taken together, high-glucose environment reduces the expression of TM and TLR4 in keratinocytes possibly through the action of TNF-α, and recombinant sTM can increase the TLR4 expression and promote wound healing under diabetic condition.
Assuntos
Complicações do Diabetes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Trombomodulina/fisiologia , Receptor 4 Toll-Like/metabolismo , Cicatrização , Animais , Linhagem Celular Tumoral , Deleção de Genes , Regulação da Expressão Gênica , Glucose/química , Humanos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Queratinócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Recombinantes/metabolismo , Pele/metabolismo , Estreptozocina/química , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The membrane glycoprotein thrombomodulin (TM) has been implicated in keratinocyte differentiation and wound healing, but its specific function remains undetermined. The epidermis-specific TM knockout mice were generated to investigate the function of TM in these biological processes. Primary cultured keratinocytes obtained from TM(lox/lox); K5-Cre mice, in which TM expression was abrogated, underwent abnormal differentiation in response to calcium induction. Poor epidermal differentiation, as evidenced by downregulation of the terminal differentiation markers loricrin and filaggrin, was observed in TM(lox/lox); K5-Cre mice. Silencing TM expression in human epithelial cells impaired calcium-induced extracellular signal-regulated kinase pathway activation and subsequent keratinocyte differentiation. Compared with wild-type mice, the cell spreading area and wound closure rate were lower in keratinocytes from TM(lox/lox); K5-Cre mice. In addition, the lower density of neovascularization and smaller area of hyperproliferative epithelium contributed to slower wound healing in TM(lox/lox); K5-Cre mice than in wild-type mice. Local administration of recombinant TM (rTM) accelerated healing rates in the TM-null skin. These data suggest that TM has a critical role in skin differentiation and wound healing. Furthermore, rTM may hold therapeutic potential for the treatment of nonhealing chronic wounds.
Assuntos
Queratinócitos/citologia , Queratinócitos/fisiologia , Trombomodulina/genética , Trombomodulina/metabolismo , Cicatrização/fisiologia , Animais , Cálcio/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular , Movimento Celular/fisiologia , Células Epidérmicas , Epiderme/fisiologia , Proteínas Filagrinas , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Camundongos Knockout , Neovascularização Fisiológica/fisiologia , Fosforilação/fisiologia , Cultura Primária de CélulasRESUMO
Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.
Assuntos
Fator de Crescimento Epidérmico/metabolismo , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/genética , Receptores de Complemento/biossíntese , Receptores de Complemento/genética , Animais , Movimento Celular , Proliferação de Células , Colágeno/química , Combinação de Medicamentos , Células Endoteliais/citologia , Endotélio Vascular/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Laminina/química , Camundongos , Neovascularização Patológica , Oxigênio/química , Oxigênio/metabolismo , Estrutura Terciária de Proteína , Proteoglicanas/química , Degeneração Retiniana/patologia , Transdução de SinaisRESUMO
The expression of thrombomodulin (TM), a membrane glycoprotein, is upregulated in neoepidermis during cutaneous wound healing. Rhomboid-like-2 (RHBDL2), an intramembrane serine protease, specifically cleaves TM at the transmembrane domain and causes the release of soluble TM (sTM). However, the physiological functions of TM and RHBDL2 in wound healing remain unclear. We demonstrated that both TM and RHBDL2 are upregulated in HaCaT cells stimulated by scratch wounds; furthermore, increased sTM was found in culture medium. Conversely, inhibition of RHBDL2 by 3,4-dichloroisocoumarin (DCI) or short hairpin RNA significantly inhibited wound-induced TM ectodomain shedding and wound healing. Both conditioned media from multiple-scratch-wounded HaCaT and recombinant sTM accelerated wound healing in HaCaT cells; such effects were abrogated by anti-TM antibodies. The RNA released from injured cells is involved in the induction of TM and RHBDL2. RHBDL2 and sTM were upregulated in ex vivo tissue culture of the injured skin. Furthermore, DCI inhibited sTM production and wound healing; this was reversed by recombinant sTM in mice. Thus, RHBDL2 and TM have important roles in wound healing via the release of sTM from keratinocytes; this may function as an autocrine/paracrine signal promoting wound healing.