RESUMEN
RATIONALE: Sialylation by α2,3-sialyltransferases has been shown to be a crucial glycosylation step in the generation of functional selectin ligands. Recent evidence suggests that sialylation also affects the binding of chemokines to their corresponding receptor. OBJECTIVE: Because the chemokine receptors for Ccl5 and Ccl2 are important in atherogenic recruitment of neutrophils and monocytes, we here investigated the role of α2,3-sialyltransferase IV (ST3Gal-IV) in Ccl5- and Ccl2-mediated myeloid cell arrest and further studied its relevance in a mouse model of atherosclerosis. METHODS AND RESULTS: St3Gal4-deficient myeloid cells showed a reduced binding of Ccl5 and an impaired Ccl5-triggered integrin activation. Correspondingly, Ccl5-induced arrest on tumor necrosis factor-α-stimulated endothelium was almost completely abrogated, as observed in flow chamber adhesion assays and during ex vivo perfusion or intravital microscopy of carotid arteries. Moreover, Ccl5-triggered neutrophil and monocyte extravasation into the peritoneal cavity was severely reduced in St3Gal4(-/-) mice. In contrast, St3Gal4 deficiency did not significantly affect Ccl2 binding and only marginally decreased Ccl2-induced flow arrest of myeloid cells. In agreement with the crucial role of leukocyte accumulation in atherogenesis, and the importance of Ccl5 chemokine receptors mediating myeloid cell recruitment to atherosclerotic vessels, St3Gal4 deficiency drastically reduced the size, stage, and inflammatory cell content of atherosclerotic lesions in Apoe(-/-) mice on high-fat diet. CONCLUSIONS: In summary, these findings identify ST3Gal-IV as a promising target to reduce inflammatory leukocyte recruitment and arrest.
Asunto(s)
Aterosclerosis/enzimología , Quimiocina CCL5/fisiología , Rodamiento de Leucocito/fisiología , Células Mieloides/patología , Sialiltransferasas/deficiencia , Animales , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/patología , Aterosclerosis/prevención & control , Quimiocina CCL2/metabolismo , Grasas de la Dieta/toxicidad , Femenino , Inflamación , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/farmacología , Procesamiento Proteico-Postraduccional , Sialiltransferasas/genética , Sialiltransferasas/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Molécula 1 de Adhesión Celular Vascular/metabolismo , beta-Galactosida alfa-2,3-SialiltransferasaRESUMEN
AIMS: The SDF-1α/CXCR4 dyad was previously shown by us and others to be instrumental in intimal hyperplasia as well as early stage atherosclerosis. We here sought to investigate its impact on clinically relevant stages of atherosclerosis in mouse and man. METHODS AND RESULTS: Immunohistochemical analysis of CXCR4 expression in human atherosclerotic lesions revealed a progressive accumulation of CXCR4(+) cells during plaque progression. To address causal involvement of CXCR4 in advanced stages of atherosclerosis we reconstituted LDLr(-/-) mice with autologous bone marrow infected with lentivirus encoding SDF-1α antagonist or CXCR4 degrakine, which effects proteasomal degradation of CXCR4. Functional CXCR4 blockade led to progressive plaque expansion with disease progression, while also promoting intraplaque haemorrhage. Moreover, CXCR4 knockdown was seen to augment endothelial adhesion of neutrophils. Concordant with this finding, inhibition of CXCR4 function increased adhesive capacity and reduced apoptosis of neutrophils and resulted in hyperactivation of circulating neutrophils. Compatible with a role of the neutrophil CXCR4 in end-stage atherosclerosis, CXCR4 expression by circulating neutrophils was lowered in patients with acute cardiovascular syndromes. CONCLUSION: In conclusion, CXCR4 contributes to later stages of plaque progression by perturbing neutrophil function.
Asunto(s)
Aterosclerosis/genética , Hemorragia/genética , Neutrófilos/metabolismo , Placa Aterosclerótica/genética , Receptores CXCR4/genética , Animales , Aterosclerosis/metabolismo , Aterosclerosis/patología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Trasplante de Médula Ósea , Adhesión Celular , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Progresión de la Enfermedad , Células Endoteliales/metabolismo , Células Endoteliales/patología , Femenino , Regulación de la Expresión Génica , Vectores Genéticos , Hemorragia/metabolismo , Hemorragia/patología , Humanos , Lentivirus/genética , Lentivirus/metabolismo , Ratones , Ratones Noqueados , Neutrófilos/patología , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores CXCR4/antagonistas & inhibidores , Receptores CXCR4/metabolismo , Receptores de LDL/deficiencia , Receptores de LDL/genética , Transducción de SeñalRESUMEN
Myocardial infarction (MI) induces a complex inflammatory immune response, followed by the remodelling of the heart muscle and scar formation. The rapid regeneration of the blood vessel network system by the attraction of hematopoietic stem cells is beneficial for heart function. Despite the important role of chemokines in these processes, their use in clinical practice has so far been limited by their limited availability over a long time-span in vivo. Here, a method is presented to increase physiological availability of chemokines at the site of injury over a defined time-span and simultaneously control their release using biodegradable hydrogels. Two different biodegradable hydrogels were implemented, a fast degradable hydrogel (FDH) for delivering Met-CCL5 over 24 hrs and a slow degradable hydrogel (SDH) for a gradual release of protease-resistant CXCL12 (S4V) over 4 weeks. We demonstrate that the time-controlled release using Met-CCL5-FDH and CXCL12 (S4V)-SDH suppressed initial neutrophil infiltration, promoted neovascularization and reduced apoptosis in the infarcted myocardium. Thus, we were able to significantly preserve the cardiac function after MI. This study demonstrates that time-controlled, biopolymer-mediated delivery of chemokines represents a novel and feasible strategy to support the endogenous reparatory mechanisms after MI and may compliment cell-based therapies.
Asunto(s)
Materiales Biocompatibles/química , Quimiocinas/uso terapéutico , Hidrogeles/química , Infarto del Miocardio/tratamiento farmacológico , Miocardio/metabolismo , Ingeniería de Proteínas , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Quimiocinas/farmacología , Pruebas de Función Cardíaca , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Infarto del Miocardio/diagnóstico por imagen , Infarto del Miocardio/patología , Infarto del Miocardio/fisiopatología , Neovascularización Fisiológica , Infiltración Neutrófila , UltrasonografíaRESUMEN
OBJECTIVE: The chemokine receptor CX(3)CR1 is an inflammatory mediator in vascular diseases. On platelets, its ligation with fractalkine (CX(3)CL1) induces platelet activation followed by leukocyte recruitment to activated endothelium. Here, we evaluated the expression and role of platelet-CX(3)CR1 during hyperlipidemia and vascular injury. METHODS AND RESULTS: The existence of CX(3)CR1 on platelets at mRNA and protein level was analyzed by RT-PCR, quantitative (q)PCR, FACS analysis, and Western blot. Elevated CX(3)CR1 expression was detected on human platelets after activation and, along with increased binding of CX(3)CL1, platelet CX(3)CR1 was also involved in the formation of platelet-monocyte complexes. Interestingly, the expression of CX(3)CR1 was elevated on platelets from hyperlipidemic mice. Accordingly, CX(3)CL1-binding and the number of circulating platelet-monocyte complexes were increased. In addition, CX(3)CR1 supported monocyte arrest on inflamed smooth muscle cells in vitro, whereas CX(3)CR1-deficient platelets showed decreased adhesion to the denuded vessel wall in vivo. CONCLUSIONS: Platelets in hyperlipidemic mice display increased CX(3)CR1-expression and assemble with circulating monocytes. The formation of platelet-monocyte complexes and the detection of platelet-bound CX(3)CL1 on inflamed smooth muscle cells suggest a significant involvement of the CX(3)CL1-CX(3)CR1 axis in platelet accumulation and monocyte recruitment at sites of arterial injury in atherosclerosis.
Asunto(s)
Plaquetas/metabolismo , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Hiperlipidemias/genética , Monocitos/metabolismo , ARN Mensajero/genética , Receptores de Citocinas/genética , Receptores del VIH/genética , Animales , Aterosclerosis/genética , Aterosclerosis/metabolismo , Aterosclerosis/patología , Western Blotting , Receptor 1 de Quimiocinas CX3C , Línea Celular , Modelos Animales de Enfermedad , Endotelio Vascular/patología , Citometría de Flujo , VIH-2 , Humanos , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Activación Plaquetaria , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores de Citocinas/biosíntesis , Receptores del VIH/biosíntesisRESUMEN
It is generally accepted that G-protein coupled receptors (GPCR), like chemokine receptors, form dimers or higher order oligomers. Such homo- and heterophilic interactions have been identified not only among and between chemokine receptors of CC- or CXC-subfamilies, but also between chemokine receptors and other classes of GPCR, like the opioid receptors. Oligomerization affects different aspects of receptor physiology, like ligand affinity, signal transduction and the mode of internalization, in turn influencing physiologic processes such as cell activation and migration. As particular chemokine receptor pairs exert specific modulating effects on their individual functions, they might play particular roles in various disease types, such as cancer. Hence, chemokine receptor heteromers might represent attractive therapeutic targets. This review highlights the state-of-the-art knowledge on the technical and functional aspects of chemokine receptor multimerization in chemokine signaling and biology.
Asunto(s)
Quimiocinas/metabolismo , Receptores de Quimiocina/metabolismo , Transducción de Señal , Animales , Quimiocinas/química , Humanos , Receptores de Quimiocina/químicaRESUMEN
The chemokine CCL5 recruits monocytes into inflamed tissues by triggering primarily CCR1-mediated arrest on endothelial cells, whereas subsequent spreading is dominated by CCR5. The CCL5-induced arrest can be enhanced by heteromer formation with CXCL4. To identify mechanisms for receptor-specific functions, we employed CCL5 mutants and transfectants expressing receptor chimeras carrying transposed extracellular regions. Mutation of the basic 50s cluster of CCL5, a coordinative site for CCL5 surface presentation, reduced CCR5- but not CCR1-mediated arrest and transmigration. Impaired arrest was restored by exchanging the CCR5-N-terminus for that of CCR1, which supported arrest even without the 50s cluster, whereas mutation of the basic 40s cluster essential for proteoglycan binding of CCL5 could not be rescued. The enhancement of CCL5-induced arrest by CXCL4 was mediated by CCR1 requiring its third extracellular loop. The domain exchanges did not affect formation and co-localisation of receptor dimers, indicating a sensing role of the third extracellular loop for hetero-oligomers in an arrest microenvironment. Our data identify confined targetable regions of CCR1 specialised to facilitate CCL5-induced arrest and enhanced responsiveness to the CXCL4-CCL5 heteromer.
Asunto(s)
Proteínas Serina-Treonina Quinasas/metabolismo , Receptores CCR5/metabolismo , Animales , Movimiento Celular/genética , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Barajamiento de ADN/métodos , Dimerización , Células HEK293 , Humanos , Ratones , Mutación/genética , Factor Plaquetario 4/genética , Factor Plaquetario 4/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína/genética , Receptores CCR5/genética , Transgenes/genéticaRESUMEN
Atherosclerosis is characterized by chronic inflammation of the arterial wall due to chemokine-driven mononuclear cell recruitment. Activated platelets can synergize with chemokines to exacerbate atherogenesis; for example, by deposition of the chemokines platelet factor-4 (PF4, also known as CXCL4) and RANTES (CCL5), triggering monocyte arrest on inflamed endothelium. Homo-oligomerization is required for the recruitment functions of CCL5, and chemokine heteromerization has more recently emerged as an additional regulatory mechanism, as evidenced by a mutual modulation of CXCL8 and CXCL4 activities and by enhanced monocyte arrest resulting from CCL5-CXCL4 interactions. The CCL5 antagonist Met-RANTES reduces diet-induced atherosclerosis; however, CCL5 antagonism may not be therapeutically feasible, as suggested by studies using Ccl5-deficient mice which imply that direct CCL5 blockade would severely compromise systemic immune responses, delay macrophage-mediated viral clearance and impair normal T cell functions. Here we determined structural features of CCL5-CXCL4 heteromers and designed stable peptide inhibitors that specifically disrupt proinflammatory CCL5-CXCL4 interactions, thereby attenuating monocyte recruitment and reducing atherosclerosis without the aforementioned side effects. These results establish the in vivo relevance of chemokine heteromers and show the potential of targeting heteromer formation to achieve therapeutic effects.