RESUMEN
At least seven distinct epidermal growth factor (EGF) ligands bind to and activate the EGF receptor (EGFR). This activation plays an important role in the embryo and in the maintenance of adult tissues. Importantly, pharmacologic EGFR inhibition also plays a critical role in the pathophysiology of diverse disease states, especially cancer. The roles of specific EGFR ligands are poorly defined in these disease states. Accumulating evidence suggests a role for transforming growth factor α (TGFα) in skin, lung, and kidney disease. To explore the role of Tgfa, we generated a monoclonal antibody (mAb41) that binds to and neutralizes human Tgfa with high affinity (KD = 36.5 pM). The antibody also binds human epiregulin (Ereg) (KD = 346.6 pM) and inhibits ligand induced myofibroblast cell proliferation (IC50 values of 0.52 and 1.12 nM for human Tgfa and Ereg, respectively). In vivo, a single administration of the antibody to pregnant mice (30 mg/kg s.c. at day 14 after plug) or weekly administration to neonate mice (20 mg/kg s.c. for 4 weeks) phenocopy Tgfa knockout mice with curly whiskers, stunted growth, and expansion of the hypertrophic zone of growth plate cartilage. Humanization of this monoclonal antibody to a human IgG4 antibody (LY3016859) enables clinical development. Importantly, administration of the humanized antibody to cynomolgus monkeys is absent of the skin toxicity observed with current EGFR inhibitors used clinically and no other pathologies were noted, indicating that neutralization of Tgfa could provide a relatively safe profile as it advances in clinical development.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Factor de Crecimiento Transformador alfa/metabolismo , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Anticuerpos Monoclonales Humanizados/metabolismo , Anticuerpos Monoclonales Humanizados/farmacocinética , Anticuerpos Neutralizantes/metabolismo , Anticuerpos Neutralizantes/farmacología , Línea Celular , Proliferación Celular/efectos de los fármacos , Epirregulina , Humanos , Inmunoglobulina G/inmunología , Macaca fascicularis , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Datos de Secuencia Molecular , Miofibroblastos/citología , Miofibroblastos/efectos de los fármacos , Miofibroblastos/metabolismo , Unión Proteica , Factor de Crecimiento Transformador alfa/genéticaRESUMEN
OBJECTIVE: Similarities between neovascular ingrowth in atherosclerotic plaques and angiogenesis in tumors suggest that antiangiogenic factors that target tumor expansion may prove efficacious in the treatment of atherosclerosis. This study examined whether an oral DNA vaccine against the murine VEGF receptor 2 (Flk-1) with demonstrated antitumor effect through inhibition of pathological neovascularization can prevent or retard progression of atherosclerosis in hyperlipidemic low density lipoprotein receptor-deficient (LDLr-/-) mice. METHODS AND RESULTS: Vaccination against Flk-1 resulted in T cell activation, suppression of neoangiogenesis, and a marked reduction in atherosclerosis which was independent of hypercholesterolemia in both male and female mice. Immunohistochemical characterization of aortic sinus lesions showed that the decreased lesion area was not associated with reduced plaque stability and had a lower density of microvessels. CONCLUSIONS: These findings demonstrate for the first time that a DNA vaccine targeting activated endothelial cells in atherosclerotic lesions provides direct atheroprotective effects.
Asunto(s)
Aterosclerosis/terapia , Receptores de LDL/deficiencia , Vacunación/métodos , Vacunas de ADN/uso terapéutico , Receptor 2 de Factores de Crecimiento Endotelial Vascular/efectos de los fármacos , Animales , Aterosclerosis/inmunología , Aterosclerosis/metabolismo , Modelos Animales de Enfermedad , Ensayo de Inmunoadsorción Enzimática , Femenino , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos C57BL , Linfocitos T/inmunología , Resultado del Tratamiento , Receptor 2 de Factores de Crecimiento Endotelial Vascular/sangreRESUMEN
Vasculogenic mimicry (VM), the formation of matrix-rich vascular-like networks in three-dimensional culture corresponding with the expression of vascular cell-associated genes, and the lining of matrix-rich networks in situ, has been observed in highly aggressive and malignant melanoma. However, little is known about the molecular underpinnings of this phenomenon. On the basis of gene profiling, protein detection, and immunohistochemistry, aggressive relative to poorly aggressive melanoma showed up-regulation of tissue factor (TF), TF pathway inhibitor 1 (TFPI-1) and 2 (TFPI-2), critical genes that initiate and regulate the coagulation pathways. The procoagulant function of TF on highly aggressive melanoma is shown to be regulated by TFPI-1 but not by TFPI-2. Thus, aggressive melanoma exhibits endothelial cell-like anticoagulant mechanisms that may contribute to the fluid-conducting potential of melanoma cell-lined networks, as studied by correlative in vivo Doppler flow measurements. Antibody inhibition experiments reveal that TFPI-2 is required for VM in vitro, but plasmin is an unlikely target protease of TFPI-2. Blockade of TFPI-2 suppressed matrix metalloproteinase-2 activation, and, therefore, TFPI-2 appears to regulate an essential pathway of VM. TFPI-2 is synthesized by endothelial and tumor cells, which deposit TFPI-2 into extracellular matrices. Culturing poorly aggressive melanoma cells on three-dimensional matrix containing recombinant TFPI-2 produces some of the phenotypic changes associated with aggressive, vasculogenic melanoma cells. Thus, TFPI-2 contributes to VM plasticity, whereas TFPI-1 has anticoagulant functions of relevance for perfusion of VM channels formed by TF-expressing melanoma cells.
Asunto(s)
Glicoproteínas/fisiología , Lipoproteínas/fisiología , Melanoma/irrigación sanguínea , Animales , Células CHO , Cricetinae , Glicoproteínas/biosíntesis , Glicoproteínas/genética , Humanos , Lipoproteínas/biosíntesis , Lipoproteínas/genética , Melanoma/genética , Melanoma/metabolismo , Melanoma/patología , Ratones , Ratones Desnudos , Neovascularización Patológica/genética , Neovascularización Patológica/metabolismo , Neoplasias Cutáneas/irrigación sanguínea , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Tromboplastina/antagonistas & inhibidores , Tromboplastina/fisiología , Trasplante Heterólogo , Neoplasias de la Úvea/irrigación sanguínea , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Neoplasias de la Úvea/patologíaRESUMEN
Factor X is activated to factor Xa (fXa) in the extrinsic coagulation pathway by the tissue factor (TF)/factor VIIa (fVIIa) complex. Upon activation, the fXa molecule remains associated with the TF/fVIIa complex, and this ternary complex is known to activate protease-activated receptors (PARs) 1 and 2. Activation of fVII in the TF complex by fXa is also seen at physiologic concentrations. The ternary complexes TF/fVII/fXa, TF/fVIIa/fX, and TF/fVIIa/fXa are therefore all physiologically relevant and of interest as targets for inhibition of both coagulation and cell-signaling pathways that are important in cardiovascular disease and inflammation. We therefore present a model of the TF/fVIIa/fXa complex, built with the use of the available structures of the TF/fVIIa complex and fXa by protein-protein docking calculations with the program Surfdock. The fXa model has an extended conformation, similar to that of fVIIa in the TF/fVIIa complex, with extensive interactions with TF and the protease domain of fVIIa. All four domains of fXa are involved in the interaction. The gamma-carboxyglutamate (Gla) and epithelial growth factor (EGF1 and EGF2) domains of fVIIa are not significantly involved in the interaction. Docking of the Gla domain of fXa to TF/fVIIa has been reported previously. The docking results identify potential interface residues, allowing rational selection of target residues for site-directed mutagenesis. This combination of docking and mutagenesis confirms that residues Glu51 and Asn57 in the EGF1 domain, Asp92 and Asp95 in the EGF2 domain, and Asp 185a, Lys 186, and Lys134 in the protease domain of factor Xa are involved in the interaction with TF/fVIIa. Other fX protease domain residues predicted to be involved in the interaction come from the 160s loop and the N-terminus of the fX protease domain, which is oriented in such a way that activation of both fVII by fXa, and the reciprocal fX activation by fVIIa, is possible.
Asunto(s)
Factor VIIa/química , Factor Xa/química , Modelos Moleculares , Tromboplastina/química , Sitios de Unión , Dominio Catalítico , Factor de Crecimiento Epidérmico/química , Factor VIIa/metabolismo , Factor Xa/genética , Factor Xa/metabolismo , Sustancias Macromoleculares , Mutagénesis Sitio-Dirigida , Estructura Terciaria de Proteína , Tromboplastina/metabolismoRESUMEN
The anti-inflammatory effects of activated protein C (APC) have lead to its recent approval for the treatment of sepsis. Although the endothelial cell protein C receptor (EPCR) plays a crucial role in APC's protective roles in septicemia, the precise signaling mechanism of the protease APC remains unclear. In fibroblast overexpression systems, we find that APC activates protease activated receptors (PAR) 1 and 2 in an EPCR-dependent manner. Human endothelial cells (HUVECs) express PAR1, PAR2 and EPCR. Stimulation of HUVECs with either APC, or specific receptor activating peptides for PAR1 or PAR2, show that all three agonists induce a very similar set of early response genes as assessed by high density microarray analysis. Only the transcript for monocyte chemo-attractant protein-1 (MCP-1) was selectively induced by APC and the PAR1 agonist, but not by the PAR2 agonist. APC-mediated MAP kinase phosphorylation and gene induction were inhibited by cleavage blocking antibodies to PAR1, demonstrating that APC signals exclusively through PAR1 in endothelial cells. MCP-1 is protective in animal models of endotoxemia, suggesting that APC may prevent lethality in sepsis by inducing MCP-1 expression through EPCR-dependent activation of endothelial cell PAR1. These data demonstrate unexpected protective functions of the major thrombin receptor PAR1 in endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Proteína C/metabolismo , Receptor PAR-1/metabolismo , Transducción de Señal/fisiología , Clorometilcetonas de Aminoácidos/farmacología , Animales , Anticuerpos Bloqueadores/farmacología , Línea Celular , Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Receptor de Proteína C Endotelial , Endotelio Vascular/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Glicoproteínas/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Receptores de Superficie Celular , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Evidence is accumulating to suggest that TFPI-2 is involved in regulating pericellular proteases implicated in a variety of physiologic and pathologic processes including cancer cell invasion, vascular inflammation, and atherosclerosis. Recent immunohistochemical studies of advanced atherosclerotic lesions, demonstrated a similar tissue distribution for TFPI-2, High Molecular Weight Kininogen (HK), and gC1qR/p33 (gC1qR), a ubiquitously expressed, multicompartmental cellular protein involved in modulating complement, coagulation, and kinin cascades. Further studies to evaluate TFPI-2 interactions with gC1qR demonstrated direct interactions between gC1qR and TFPI-2 using immunoprecipitation and solid phase binding studies. Specific and saturable binding between TFPI-2 and gC1qR (estimated Kd: approximately 70 nM) was observed by ELISA and surface plasmon resonance (Biacore) binding assays. Binding was inhibited by antibodies to gC1qR, and was strongly dependent on the Kunitz-2 domain of TFPI-2, as deletion of this domain reduced gC1qR-TFPI-2 interactions by approximately 75%. Deletion of gC1qR amino acids 74-95, involved in C1q binding, had no effect on gC1qR binding to TFPI-2, although antibodies to this region and purified C1q both inhibited binding, most likely via allosteric effects. In contrast, HK did not affect TFPI-2 binding to gC1qR. Binding of TFPI-2 to gC1qR produced statistically significant but modest reductions in TFPI-2 inhibition of plasmin, but had no effect on kallikrein inhibition in fluid phase chromogenic assays. Taken together, these data suggest that gC1qR may participate in tissue remodeling and inflammation by localizing TFPI-2 to the pericellular environment to modulate local protease activity and regulate HK activation.
Asunto(s)
Endotelio Vascular/patología , Glicoproteínas/metabolismo , Receptores de Hialuranos/metabolismo , Inflamación/etiología , Proteínas Portadoras , Complemento C1q/farmacología , Fibrinolisina/antagonistas & inhibidores , Glicoproteínas/farmacología , Humanos , Receptores de Hialuranos/farmacología , Calicreínas/antagonistas & inhibidores , Quininógeno de Alto Peso Molecular/farmacología , Proteínas Mitocondriales , Unión Proteica/efectos de los fármacosRESUMEN
Lyst(beige) mice crossed with hyperlipidemic low density lipoprotein receptor-deficient mice (BgLDLr(-/-)) display increased lesion area and a more stable lesion morphology. To verify that the beige phenotype is not unique to LDLr(-/-) mice, we examined atherosclerosis in beige, apolipoprotein E-deficient mutant mice (BgApoE(-/-)). Severe diet-induced hyperlipidemia in BgApoE(-/-) mice resulted in increased aortic sinus lesion areas compared with controls. Minimal aortic lesions were observed in both genotypes on a chow diet. Nevertheless, BgApoE(-/-) mice displayed drastically reduced aortic sinus lesion growth. Reconstitution with bone marrow (BM) from green fluorescent protein mice created chimeric animals that allowed for the identification of donor-derived cells within lesions. Expressing the beige mutation exclusively in BM-derived cells had no impact on plaque development, yet the beige mutation in all cells except the BM-derived cells led to significantly larger aortic sinus lesion areas. Both mRNA and secreted protein levels of monocyte chemoattractant protein 1 were altered in quiescent and phorbol ester-stimulated cultured macrophages, vascular smooth muscle cells, and aortic endothelial cells isolated from BgApoE(-/-) mice. Thus, expression of the beige mutation in all cell types involved in lesion development contributed to atheroprotection in chow-fed ApoE(-/-) mice.
Asunto(s)
Alimentación Animal , Apolipoproteínas E/genética , Aterosclerosis/genética , Mutación , Proteínas/genética , Proteínas/metabolismo , Animales , Apolipoproteínas E/deficiencia , Aterosclerosis/etiología , Aterosclerosis/metabolismo , Grasas de la Dieta/administración & dosificación , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Receptores de LDL/genética , Proteínas de Transporte VesicularRESUMEN
Factor VIIa (VIIa) remains in a zymogen-like state following proteolytic activation and depends on interactions with the cofactor tissue factor (TF) for function. Val(21), Glu(154), and Met(156) are residues that are spatially close in available zymogen and enzyme structures, despite major conformational differences in the corresponding loop segments. This residue triad displays unusual side chain properties in comparison to the properties of other coagulation serine proteases. By mutagenesis, we demonstrate that these residues cooperate to stabilize the enzyme conformation and to enhance the affinity for TF. In zymogen VII, however, substitution of the triad did not change the cofactor affinity, further emphasizing the crucial role of the activation pocket in specifically stabilizing the active enzyme conformation. In comparison to VIIa(Q156), the triple mutant VIIa(N21I154Q156) had a stabilized amino-terminal Ile(16)-Asp(194) salt bridge and enhanced catalytic function. However, proteolytic and amidolytic activities of free VIIa variants were not concordantly increased. Rather, a negatively charged Asp at position 21 was the critical factor that determined whether an amidolytically more active VIIa variant also more efficiently activated the macromolecular substrate. These data thus demonstrate an unexpected complexity by which the zymogenicity-determining triad in the activation pocket of VIIa controls the active enzyme conformation and contributes to exosite interactions with the macromolecular substrate.
Asunto(s)
Precursores Enzimáticos/metabolismo , Factor VII/metabolismo , Factor VIIa/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Precursores Enzimáticos/química , Factor VII/química , Factor VIIa/química , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Resonancia por Plasmón de SuperficieRESUMEN
The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2. Thus, the prototypical thrombin receptor is the target for EPCR-dependent APC signaling, suggesting a role for this receptor cascade in protection from sepsis.