RESUMEN
The potential role of alpha 5 beta 1 (VLA-5) in leukocyte trafficking in zymosan-induced acute peritonitis was determined. In naïve mice, approximately 98% of Gr1(high) cells (PMN) in bone marrow and circulation were alpha 5 beta 1-negative; these profiles were modestly affected by peritoneal injection of zymosan. In contrast, approximately 30% of Gr1(high) cells recruited by zymosan (24 h) to the peritoneal cavity expressed alpha 5 beta 1. With respect to F4/80(+) cells, approximately 60% of bone marrow and peripheral blood populations expressed alpha 5 beta 1, with approximately 90% positivity in resident cells of noninflamed peritoneum. Analysis of alpha 5 beta 1 expression revealed inflammation-dependent increased expression on Gr1(high) and F4/80(+) cells in bone marrow, blood, and peritoneal cavity. Blockade of alpha 5 beta 1, by an anti-alpha 5 mAb, attenuated zymosan-induced 24 h recruitment of Gr1(high) and F4/80(+) cells. At least one underlying mechanism of this action was reduction of cell adhesion and transmigration across microvascular vessels, as revealed by intravital microscopy. Confocal analyses indicated that deposition of fibronectin, the principal ligand for alpha 5 beta 1, was up-regulated significantly on and around the inflamed mesenteric microvasculature. These data suggest that the effects of alpha 5-blockade may be a result of inhibition of alpha 5 beta 1-dependent leukocyte adhesion to and migration along the fibronectin matrix. This is the first report that identifies a functional role for alpha 5 beta 1 in leukocyte trafficking during acute inflammation.
Asunto(s)
Quimiotaxis de Leucocito/inmunología , Integrina alfa5beta1/inmunología , Leucocitos/inmunología , Peritonitis/inmunología , Animales , Adhesión Celular , Fibronectinas/inmunología , Fibronectinas/metabolismo , Integrina alfa5beta1/metabolismo , Leucocitos/metabolismo , Masculino , Ratones , Microscopía Confocal , Peritonitis/metabolismoRESUMEN
Recently, a new class of selective integrin alpha5beta1inhibitors consisting of a heterocyclic based scaffold was published. Herein the SAR and pharmacokinetic profiles of N-phenyl piperidine derivatives are described.
Asunto(s)
Aminopiridinas/química , Integrina alfa5beta1/antagonistas & inhibidores , Fenilalanina/análogos & derivados , Piperidinas/química , Administración Oral , Aminopiridinas/síntesis química , Aminopiridinas/farmacocinética , Animales , Perros , Diseño de Fármacos , Integrina alfa5beta1/metabolismo , Masculino , Fenilalanina/síntesis química , Fenilalanina/química , Fenilalanina/farmacocinética , Piperidinas/síntesis química , Piperidinas/farmacocinética , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Previous research within our laboratories identified the 3-hydroxypyrrolidine scaffold 1 as a new and selective integrin alpha5beta1 inhibitor class which was designed for local administration. Herein the discovery of new orally available integrin alpha5beta1 inhibitor scaffolds for potential systemic treatment is described.
Asunto(s)
Integrina alfa5beta1/antagonistas & inhibidores , Pirrolidinas/química , Administración Oral , Animales , Diseño de Fármacos , Semivida , Integrina alfa5beta1/metabolismo , Masculino , Pirrolidinas/síntesis química , Pirrolidinas/farmacocinética , Ratas , Ratas Wistar , Relación Estructura-ActividadRESUMEN
PURPOSE: The aim of this study was to determine the expression and localization of integrin alpha5beta1 in human retinal pigment epithelium (RPE) and its ability to modulate RPE cell attachment, proliferation, migration, and F-actin cytoskeleton distribution. METHODS: Expression and localization of alpha5beta1 were analyzed on human RPE by immunoblot/immunofluorescence. Polarized secretion of fibronectin was measured. RPE attachments to different substrates were determined using cell attachment screening kits. BrdU incorporation and wound-healing assays were used to test hfRPE proliferation and migration. F-actin cytoskeleton was visualized with phalloidin. RESULTS: Integrin alpha5beta1 was detected in native adult and fetal human RPE. The alpha5-subunit is predominantly localized at the apical membrane of hfRPE, whereas the beta1-subunit is uniformly detected at the apical/basolateral membranes. The authors also found that hfRPE cultures secrete significant amounts of fibronectin to the apical bath. JSM6427, a specific integrin alpha5beta1 antagonist, significantly inhibited hfRPE cell attachment to fibronectin, but not laminin, or collagen I or IV. JSM6427 also showed a strong inhibitory effect on bFGF, PDGF-BB, and serum-induced cell migration and proliferation. Furthermore, JSM6427 induced significant disruption of the F-actin cytoskeleton of dividing RPE cells but had no effect on quiescent cells. CONCLUSIONS: The apical localization of alpha5beta1 and the secretion of fibronectin to the apical bath suggest the presence of an autocrine loop that can guide the migration of RPE. The strong inhibitory effects of JSM6427 on human RPE cell attachment, proliferation, and migration is probably mediated by F-actin cytoskeletal disruption in proliferating cells and suggests a potential clinical use of this compound in proliferative retinopathies.
Asunto(s)
Adhesión Celular/fisiología , Movimiento Celular/fisiología , Proliferación Celular , Integrina alfa5beta1/fisiología , Enfermedades de la Retina/metabolismo , Epitelio Pigmentado de la Retina/citología , Actinas/metabolismo , Adhesión Celular/efectos de los fármacos , Membrana Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Fibronectinas/metabolismo , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Integrina alfa5beta1/antagonistas & inhibidores , Propionatos/farmacología , Piridinas/farmacología , Pirrolidinas/farmacología , Epitelio Pigmentado de la Retina/metabolismo , Cicatrización de Heridas/fisiologíaRESUMEN
The integrin alpha5beta1 has been previously implicated in tumor angiogenesis, but its role in the remodeling of both blood vessels and lymphatics during inflammation is at an early stage of understanding. We examined this issue using a selective, small-molecule inhibitor of alpha5beta1 integrin, 2-aroylamino-3-{4-[(pyridin-2-ylaminomethyl)heterocyclyl]phenyl}propionic acid (JSM8757), in a model of sustained airway inflammation in mice with Mycoplasma pulmonis infection, which is known to be accompanied by robust blood vessel remodeling and lymphangiogenesis. The inhibitor significantly decreased the proliferation of lymphatic endothelial cells in culture and the number of lymphatic sprouts and new lymphatics in airways of mice infected for 2 weeks but did not reduce remodeling of blood vessels in the same airways. In inflamed airways, alpha5 integrin immunoreactivity was present on lymphatic sprouts, but not on collecting lymphatics or blood vessels, and was not found on any lymphatics of normal airways. Macrophages, potential targets of the inhibitor, did not have alpha5 integrin immunoreactivity in inflamed airways. In addition, macrophage recruitment, assessed in infected airways by quantitative reverse transcription-polymerase chain reaction measurements of expression of the marker protein ionized calcium-binding adapter molecule 1 (Iba1), was not reduced by JSM8757. We conclude that inhibition of the alpha5beta1 integrin reduces lymphangiogenesis in inflamed airways after M. pulmonis infection because expression of the integrin is selectively increased on lymphatic sprouts and plays an essential role in lymphatic growth.
Asunto(s)
Linfangiogénesis/fisiología , Neumonía/metabolismo , Animales , Femenino , Inmunohistoquímica , Inflamación/metabolismo , Inflamación/microbiología , Linfangiogénesis/efectos de los fármacos , Vasos Linfáticos/efectos de los fármacos , Vasos Linfáticos/metabolismo , Vasos Linfáticos/patología , Ratones , Ratones Endogámicos C57BL , Infecciones por Mycoplasma/metabolismo , Infecciones por Mycoplasma/fisiopatología , Mycoplasma pulmonis , Neumonía/microbiología , Propionatos/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Starting from the structure of integrin alphavbeta3 in a complex with a peptidic ligand plus SAR data on nonpeptidic ligands, we derived a new class of integrin alpha5beta1 antagonists (1). Several synthesis strategies were applied to evaluate the chemical space around the essential pharmacophore groups R1 to R3 to obtain highly active and selective pyrrolidine derivatives as integrin alpha5beta1 antagonists. Integrin selectivity was controlled by switching from a sulfonamide moiety to a mesitylene amide moiety for R3. This finding represents a general feature for modulating selectivity toward other related integrin receptors. On the basis of the encouraging results from various in vitro studies, the most active compounds were selected for further in vivo studies in animal models and preclinical development.
Asunto(s)
Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/química , Piridinas/síntesis química , Pirrolidinas/síntesis química , Diseño de Fármacos , Ésteres , Integrina alfaVbeta3/química , Ligandos , Modelos Moleculares , Péptidos Cíclicos/química , Conformación Proteica , Piridinas/química , Pirrolidinas/química , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Sulfonamidas/químicaRESUMEN
Integrins are transmembrane receptor proteins critical for growth and stabilization of vessels, but the mechanisms by which integrin activities are involved in neoangiogenesis of the eye remain unclear. Specific inhibitors to fibronectin receptor integrin alpha(5)beta(1) impeded pathological neovascularization in vivo. Our objective was to determine whether alpha(5)beta(1) plays a role in ocular angiogenesis, and whether a novel alpha(5)beta(1)-inhibiting small molecule is able to reduce angiogenesis in a model of inflammatory corneal neovascularization. Corneal neovascularization was induced in C57Bl/6 mice by NaOH-application and debridement of the limbal epithelium. Mice were randomized into six groups receiving either no treatment, or intraperitoneal osmotic pumps delivering three different doses of integrin antagonist or control substance on day 10 after scraping. In order to quantify the neovascular response, flatmounts were stained with FITC-CD31. Integrin alpha(5) expression was determined by immunohistochemistry and quantified by semiquantitative western blot analysis. Influence of integrin antagonist treatment on the mRNA expression of VEGF, bFGF and integrin alpha(5) was quantified by real-time RT-PCR. Vascularized corneas demonstrated a strong up-regulation of integrin alpha(5) within affected areas. Animals treated systemically with alpha(5)beta(1)-inhibiting small molecule showed a significant inhibition and regression of corneal neovascularization. PCR analysis evinced a significant up-regulation of VEGF and integrin alpha(5) mRNA levels in injured animals compared to controls, and a significant reduction of integrin alpha(5) mRNA in substance-treated animals compared to control substance, but no significant differences of bFGF levels in all groups. Western blot analysis of integrin alpha(5)beta(1) protein expression showed a trend towards up-regulation in injured animals, both control substance-treated and those treated with the alpha(5)beta(1)-inhibiting small molecule. Systemic delivery of an alpha(5)beta(1)-inhibiting small molecule inhibits and regresses corneal neovascularization induced by mechanical-alkali burn corneal injury. These results suggest an essential role for the integrin alpha(5)beta(1) in pathological neovascular processes of the cornea. Integrin alpha(5)beta(1) inhibitors could become a new approach for treatment of neovascularization in the eye.
Asunto(s)
Neovascularización de la Córnea/fisiopatología , Integrina alfa5beta1/fisiología , Inhibidores de la Angiogénesis/sangre , Inhibidores de la Angiogénesis/farmacología , Inhibidores de la Angiogénesis/uso terapéutico , Animales , Western Blotting , Córnea/metabolismo , Neovascularización de la Córnea/inducido químicamente , Neovascularización de la Córnea/tratamiento farmacológico , Neovascularización de la Córnea/prevención & control , Relación Dosis-Respuesta a Droga , Femenino , Integrina alfa5beta1/antagonistas & inhibidores , Integrina alfa5beta1/metabolismo , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa/métodos , Pirrolidinas/sangre , Pirrolidinas/farmacología , Pirrolidinas/uso terapéutico , Hidróxido de SodioRESUMEN
Integrin alpha(5)beta(1) plays an important role in developmental angiogenesis, but its role in various types of pathologic neovascularization has not been completely defined. In this study, we found strong up-regulation of alpha(5)beta(1) in choroidal neovascularization. Implantation of an osmotic pump delivering 1.5 or 10 microg/h ( approximately 1.8 or 12 mg/kg/day) of 3-(2-{1-alkyl-5-[(pyridin-2-ylamino)-methyl]-pyrrolidin-3-yloxy}-acetylamino)-2-(alkylamino)-propionic acid (JSM6427), a selective alpha(5)beta(1) antagonist, caused significant suppression of choroidal neovascularization; the area of neovascularization was reduced by 33 to 40%. When an osmotic pump delivering 10 microg/h of JSM6427 was implanted 7 days after rupture of Bruch's membrane, there was terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining in vascular cells within the neovascularization and significant regression of the neovascularization over the next week. JSM6427 also induced apoptosis of cultured vascular endothelial cells. Fibronectin stimulates phosphorylation of extracellular signal-regulated kinase (ERK) in alpha(5)beta(1)-expressing cells that is blocked by JSM6427. These data suggest that alpha(5)beta(1) plays a role in the development and maintenance of choroidal neovascularization and provides a target for therapeutic intervention.