RESUMO
The Ectonucleotide Pyrophosphatase/Phosphodiesterase 1 (ENPP1) ectoenzyme regulates vascular intimal proliferation and mineralization of bone and soft tissues. ENPP1 variants cause Generalized Arterial Calcification of Infancy (GACI), a rare genetic disorder characterized by ectopic calcification, intimal proliferation, and stenosis of large- and medium-sized arteries. ENPP1 hydrolyzes extracellular ATP to pyrophosphate (PPi) and AMP. AMP is the precursor of adenosine, which has been implicated in the control of neointimal formation. Herein, we demonstrate that an ENPP1-Fc recombinant therapeutic inhibits proliferation of vascular smooth muscle cells (VSMCs) in vitro and in vivo. Addition of ENPP1 and ATP to cultured VSMCs generated AMP, which was metabolized to adenosine. It also significantly decreased cell proliferation. AMP or adenosine alone inhibited VSMC growth. Inhibition of ecto-5'-nucleotidase CD73 decreased adenosine accumulation and suppressed the anti-proliferative effects of ENPP1/ATP. Addition of AMP increased cAMP synthesis and phosphorylation of VASP at Ser157. This AMP-mediated cAMP increase was abrogated by CD73 inhibitors or by A2aR and A2bR antagonists. Ligation of the carotid artery promoted neointimal hyperplasia in wild-type mice, which was exacerbated in ENPP1-deficient ttw/ttw mice. Prophylactic or therapeutic treatments with ENPP1 significantly reduced intimal hyperplasia not only in ttw/ttw but also in wild-type mice. These findings provide the first insight into the mechanism of the anti-proliferative effect of ENPP1 and broaden its potential therapeutic applications beyond enzyme replacement therapy.
Assuntos
5'-Nucleotidase , Adenosina , Proliferação de Células , Músculo Liso Vascular , Miócitos de Músculo Liso , Diester Fosfórico Hidrolases , Pirofosfatases , Transdução de Sinais , Diester Fosfórico Hidrolases/metabolismo , Diester Fosfórico Hidrolases/genética , Pirofosfatases/metabolismo , Pirofosfatases/genética , 5'-Nucleotidase/metabolismo , 5'-Nucleotidase/genética , Animais , Proliferação de Células/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patologia , Adenosina/metabolismo , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Miócitos de Músculo Liso/efeitos dos fármacos , Camundongos , Humanos , Monofosfato de Adenosina/metabolismo , Camundongos Endogâmicos C57BL , AMP Cíclico/metabolismo , Masculino , Calcificação Vascular/metabolismo , Calcificação Vascular/patologia , Calcificação Vascular/genéticaRESUMO
BACKGROUND AND PURPOSE: Reduced bioavailability of NO, a hallmark of sickle cell disease (SCD), contributes to intravascular inflammation, vasoconstriction, vaso-occlusion and organ damage observed in SCD patients. Soluble guanylyl cyclase (sGC) catalyses synthesis of cGMP in response to NO. cGMP-amplifying agents, including NO donors and phosphodiesterase 9 inhibitors, alleviate TNFα-induced inflammation in wild-type C57BL/6 mice and in 'humanised' mouse models of SCD. EXPERIMENTAL APPROACH: Effects of the sGC stimulator olinciguat on intravascular inflammation and renal injury were studied in acute (C57BL6 and Berkeley mice) and chronic (Townes mice) mouse models of TNFα-induced and systemic inflammation associated with SCD. KEY RESULTS: Acute treatment with olinciguat attenuated increases in plasma biomarkers of endothelial cell activation and leukocyte-endothelial cell interactions in TNFα-challenged mice. Co-treatment with hydroxyurea, an FDA-approved SCD therapeutic agent, further augmented the anti-inflammatory effect of olinciguat. In the Berkeley mouse model of TNFα-induced vaso-occlusive crisis, a single dose of olinciguat attenuated leukocyte-endothelial cell interactions, improved blood flow and prolonged survival time compared to vehicle-treated mice. In Townes SCD mice, plasma biomarkers of inflammation and endothelial cell activation were lower in olinciguat- than in vehicle-treated mice. In addition, kidney mass, water consumption, 24-h urine excretion, plasma levels of cystatin C and urinary excretion of N-acetyl-ß-d-glucosaminidase and neutrophil gelatinase-associated lipocalin were lower in Townes mice treated with olinciguat than in vehicle-treated mice. CONCLUSION AND IMPLICATIONS: Our results suggest that the sGC stimulator olinciguat attenuates inflammation, vaso-occlusion and kidney injury in mouse models of SCD and systemic inflammation.
Assuntos
Anemia Falciforme , Doenças Vasculares , Anemia Falciforme/complicações , Anemia Falciforme/tratamento farmacológico , Animais , Humanos , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Guanilil Ciclase SolúvelRESUMO
Human serum contains natural antibodies against avidin. Affinity purified natural anti-avidin human IgG exhibits affinity constants comparable to those of antibodies produced by active immunization of rabbits. Using a random hexapeptide library displayed on the filamentous M13 phage, and rabbit anti-avidin purified antibodies as a selector, we searched for epitopes shared by both selector and natural human anti-avidin IgG. This approach, enabled the isolation and identification of phagotopes bearing consensus motifs similar to sequence stretches of the avidin loops and ß-sheet regions. These phagotopes were recognized by the natural human anti-avidin antibodies. The fact that natural anti-avidin antibodies in human serum have similar epitopes to those of IgG elicited by active immunization of animals, led us to suggest that small peptide epitopes may prevent deleterious effects caused by antibodies formed against food proteins as well as therapeutic proteins.
Assuntos
Anticorpos/imunologia , Avidina/imunologia , Epitopos/imunologia , Imunoglobulina G/imunologia , Animais , Anticorpos/sangue , Ensaio de Imunoadsorção Enzimática , Mapeamento de Epitopos/métodos , Humanos , Imunoglobulina G/sangue , Biblioteca de Peptídeos , Peptídeos/imunologia , Coelhos , Especificidade da EspécieRESUMO
The transmembrane receptor guanylyl cyclase-C (GC-C), expressed on enterocytes along the intestine, is the molecular target of the GC-C agonist peptide linaclotide, an FDA-approved drug for treatment of adult patients with Irritable Bowel Syndrome with Constipation and Chronic Idiopathic Constipation. Polarized human colonic intestinal cells (T84, CaCo-2BBe) rat and human intestinal tissues were employed to examine cellular signaling and cystic fibrosis transmembrane conductance regulator (CFTR)-trafficking pathways activated by linaclotide using confocal microscopy, in vivo surface biotinylation, and protein kinase-II (PKG-II) activity assays. Expression and activity of GC-C/cGMP pathway components were determined by PCR, western blot, and cGMP assays. Fluid secretion as a marker of CFTR cell surface translocation was determined using in vivo rat intestinal loops. Linaclotide treatment (30 min) induced robust fluid secretion and translocation of CFTR from subapical compartments to the cell surface in rat intestinal loops. Similarly, linaclotide treatment (30 min) of T84 and CaCo-2BBe cells increased cell surface CFTR levels. Linaclotide-induced activation of the GC-C/cGMP/PKGII signaling pathway resulted in elevated intracellular cGMP and pVASPser239 phosphorylation. Inhibition or silencing of PKGII significantly attenuated linaclotide-induced CFTR trafficking to the apical membrane. Inhibition of protein kinase-A (PKA) also attenuated linaclotide-induced CFTR cell surface trafficking, implying cGMP-dependent cross-activation of PKA pathway. Together, these findings support linaclotide-induced activation of the GC-C/cGMP/PKG-II/CFTR pathway as the major pathway of linaclotide-mediated intestinal fluid secretion, and that linaclotide-dependent CFTR activation and recruitment/trafficking of CFTR from subapical vesicles to the cell surface is an important step in this process.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Agonistas da Guanilil Ciclase C/farmacologia , Mucosa Intestinal/metabolismo , Peptídeos/farmacologia , Transdução de Sinais , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo II/metabolismo , Humanos , Mucosa Intestinal/efeitos dos fármacos , Masculino , Transporte Proteico , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Guanilato Ciclase/metabolismoRESUMO
MRP4 mediates the efflux of cGMP and cAMP and acts as an important regulator of these secondary messengers, thereby affecting signaling events mediated by cGMP and cAMP. Immunofluorescence staining showed high MRP4 expression localized predominantly in the apical membrane of rat colonic epithelium. In vitro studies were performed using a rat colonic mucosal layer mounted in an Ussing chamber. Linaclotide activation of the guanylate cyclase-C (GC-C)/cGMP pathway induced a concentration-dependent increase in transepithelial ion current [short-circuit current (Isc)] across rat colonic mucosa (EC50: 9.2 nM). Pretreatment of colonic mucosa with the specific MRP4 inhibitor MK571 potentiated linaclotide-induced electrolyte secretion and augmented linaclotide-stimulated intracellular cGMP accumulation. Notably, pretreatment with the phosphodiesterase 5 inhibitor sildenafil increased basal Isc, but had no amplifying effect on linaclotide-induced Isc. MRP4 inhibition selectively affected the activation phase, but not the deactivation phase, of linaclotide. In contrast, incubation with a GC-C/Fc chimera binding to linaclotide abrogated linaclotide-induced Isc, returning to baseline. Furthermore, linaclotide activation of GC-C induced cGMP secretion from the apical and basolateral membranes of colonic epithelium. MRP4 inhibition blocked cGMP efflux from the apical membrane, but not the basolateral membrane. These data reveal a novel, previously unrecognized mechanism that functionally couples GC-C-induced luminal electrolyte transport and cGMP secretion to spatially restricted, compartmentalized regulation by MRP4 at the apical membrane of intestinal epithelium. These findings have important implications for gastrointestinal disorders with symptoms associated with dysregulated fluid homeostasis, such as irritable bowel syndrome with constipation, chronic idiopathic constipation, and secretory diarrhea.
Assuntos
GMP Cíclico/metabolismo , Eletrólitos/metabolismo , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Peptídeos/farmacologia , Propionatos/farmacologia , Quinolinas/farmacologia , Receptores Acoplados a Guanilato Ciclase/metabolismo , Receptores de Peptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Animais , Transporte Biológico/efeitos dos fármacos , Colo/citologia , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/fisiologia , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Feminino , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Cinética , Ratos , Ratos Sprague-Dawley , Receptores de EnterotoxinaRESUMO
The natural hormone uroguanylin regulates intestinal fluid homeostasis and bowel function through activation of guanylate cyclase-C (GC-C), resulting in increased intracellular cyclic guanosine-3',5'-monophosphate (cGMP). We report the effects of uroguanylin-mediated activation of the GC-C/cGMP pathway in vitro on extracellular cGMP transport and in vivo in rat models of inflammation- and stress-induced visceral hypersensitivity. In vitro exposure of intestinal Caco-2 cells to uroguanylin stimulated bidirectional, active extracellular transport of cGMP into luminal and basolateral spaces. cGMP transport was significantly and concentration dependently decreased by probenecid, an inhibitor of cGMP efflux pumps. In ex vivo Ussing chamber assays, uroguanylin stimulated cGMP secretion from the basolateral side of rat colonic epithelium into the submucosal space. In a rat model of trinitrobenzene sulfonic acid (TNBS)-induced visceral hypersensitivity, orally administered uroguanylin increased colonic thresholds required to elicit abdominal contractions in response to colorectal distension (CRD). Oral administration of cGMP mimicked the antihyperalgesic effects of uroguanylin, significantly decreasing TNBS- and restraint stress-induced visceromotor response to graded CRD in rats. The antihyperalgesic effects of cGMP were not associated with increased colonic spasmolytic activity, but were linked to significantly decreased firing rates of TNBS-sensitized colonic afferents in rats in response to mechanical stimuli. In conclusion, these data suggest that the continuous activation of the GC-C/cGMP pathway along the intestinal tract by the endogenous hormones guanylin and uroguanylin results in significant reduction of gastrointestinal pain. Extracellular cGMP produced on activation of GC-C is the primary mediator in this process via modulation of sensory afferent activity.
Assuntos
Guanilato Ciclase/metabolismo , Peptídeos Natriuréticos/metabolismo , Transdução de Sinais/fisiologia , Dor Visceral/metabolismo , Acetilcolina/farmacologia , Acetilglucosamina/análogos & derivados , Acetilglucosamina/farmacologia , Adenocarcinoma/patologia , Animais , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Colite/induzido quimicamente , Colite/complicações , Colo/efeitos dos fármacos , Colo/metabolismo , Neoplasias Colorretais/patologia , GMP Cíclico/análogos & derivados , GMP Cíclico/metabolismo , GMP Cíclico/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Interações Medicamentosas , Estimulação Elétrica , Feminino , Gastroenteropatias/complicações , Gastroenteropatias/etiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Hiperalgesia/fisiopatologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/fisiologia , Masculino , Mastócitos/efeitos dos fármacos , Mastócitos/metabolismo , Morfina/uso terapêutico , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Proteínas Associadas à Resistência a Múltiplos Medicamentos/metabolismo , Peptídeos Natriuréticos/uso terapêutico , Transportadores de Ânions Orgânicos Sódio-Independentes/genética , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Peroxidase/metabolismo , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Restrição Física , Ácido Trinitrobenzenossulfônico/toxicidade , Dor Visceral/tratamento farmacológico , Dor Visceral/etiologiaRESUMO
G protein-coupled receptors (GPCR) are a superfamily of receptors that are vital in a wide array of physiological processes. Modulation of GPCR signaling has been an intensive area of therapeutic study, mainly due to the diverse pathophysiological significance of GPCRs. Pepducins are cell-penetrating lipidated peptides designed to target the intracellular loops of the GPCR of interest. Pepducins can function as agonists or antagonists of their cognate receptor, making them highly useful compounds for the study of GPCR signaling. Pepducins have been used to control platelet-dependent hemostasis and thrombosis, tumor growth, invasion, and angiogenesis, as well as to improve sepsis outcomes in mice. Pepducins have been successfully designed against a wide variety of GPCRs including the protease-activated receptors (PAR1, 2, 4), the chemokine receptors (CXCR1, 2, 4), the sphingosine-1-phosphate receptor (S1P3), the adrenergic receptor (ADRA1B), and have the potential to help reveal the functions of intractable GPCRs. Pharmacokinetic, pharmacodynamic, and biodistribution studies have showed that pepducins are widely distributed throughout the body except the brain and possess appropriate drug-like properties for use in vivo. Here, we discuss the delivery, pharmacology, and biodistribution of pepducins, as well as the effects of pepducins in models of inflammation, cardiovascular disease, cancer, and angiogenesis.
Assuntos
Doença , Lipopeptídeos/farmacologia , Lipopeptídeos/farmacocinética , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Lipopeptídeos/química , Lipopeptídeos/metabolismo , Dados de Sequência Molecular , Transporte Proteico , Receptores Acoplados a Proteínas G/químicaRESUMO
The G protein-coupled receptor (GPCR), chemokine CXC-type receptor 4 (CXCR4), and its ligand, CXCL12, mediate the retention of polymorphonuclear neutrophils (PMNs) and hematopoietic stem and progenitor cells (HSPCs) in the bone marrow. Agents that disrupt CXCL12-mediated chemoattraction of CXCR4-expressing cells mobilize PMNs and HSPCs into the peripheral circulation and are therapeutically useful for HSPC collection before autologous bone marrow transplantation (ABMT). Our aim was to develop unique CXCR4-targeted therapeutics using lipopeptide GPCR modulators called pepducins. A pepducin is a synthetic molecule composed of a peptide derived from the amino acid sequence of one of the intracellular (IC) loops of a target GPCR coupled to a lipid tether. We prepared and screened a small CXCR4-targeted pepducin library and identified several pepducins with in vitro agonist activity, including ATI-2341, whose peptide sequence derives from the first IC loop. ATI-2341 induced CXCR4- and G protein-dependent signaling, receptor internalization, and chemotaxis in CXCR4-expressing cells. It also induced dose-dependent peritoneal recruitment of PMNs when administered i.p. to mice. However, when administered systemically by i.v. bolus, ATI-2341 acted as a functional antagonist and dose-dependently mediated release of PMNs from the bone marrow of both mice and cynomolgus monkeys. ATI-2341-mediated release of granulocyte/macrophage progenitor cells from the bone marrow was confirmed by colony-forming assays. We conclude that ATI-2341 is a potent and efficacious mobilizer of bone marrow PMNs and HSPCs and could represent a previously undescribed therapeutic approach for the recruitment of HSPCs before ABMT.
Assuntos
Mobilização de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/metabolismo , Peptídeos/farmacologia , Receptores CXCR4/agonistas , Transdução de Sinais/efeitos dos fármacos , Animais , Quimiotaxia/efeitos dos fármacos , Relação Dose-Resposta a Droga , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , Macaca fascicularis , Camundongos , Camundongos Endogâmicos BALB C , Receptores CXCR4/metabolismoRESUMO
Matrix metalloproteases (MMPs) play important roles in normal and pathological remodeling processes including atherothrombotic disease, inflammation, angiogenesis, and cancer. MMPs have been viewed as matrix-degrading enzymes, but recent studies have shown that they possess direct signaling capabilities. Platelets harbor several MMPs that modulate hemostatic function and platelet survival; however their mode of action remains unknown. We show that platelet MMP-1 activates protease-activated receptor-1 (PAR1) on the surface of platelets. Exposure of platelets to fibrillar collagen converts the surface-bound proMMP-1 zymogen to active MMP-1, which promotes aggregation through PAR1. Unexpectedly, MMP-1 cleaves PAR1 at a distinct site that strongly activates Rho-GTP pathways, cell shape change and motility, and MAPK signaling. Blockade of MMP1-PAR1 curtails thrombogenesis under arterial flow conditions and inhibits thrombosis in animals. These studies provide a link between matrix-dependent activation of metalloproteases and platelet-G protein signaling and identify MMP1-PAR1 as a potential target for the prevention of arterial thrombosis.