RESUMEN
We aimed to investigate the role of the CD40-CD40 ligand (CD40L) pathway in inflammation-mediated angiogenesis in proliferative diabetic retinopathy (PDR). We analyzed vitreous fluids and epiretinal fibrovascular membranes from PDR and nondiabetic patients, cultures of human retinal microvascular endothelial cells (HRMECs) and Müller glial cells and rat retinas with ELISA, immunohistochemistry, flow cytometry and Western blot analysis. Functional tests included measurement of blood-retinal barrier breakdown, in vitro angiogenesis and assessment of monocyte-HRMEC adherence. CD40L and CD40 levels were significantly increased in PDR vitreous samples. We demonstrated CD40L and CD40 expression in vascular endothelial cells, leukocytes and myofibroblasts in epiretinal membranes. Intravitreal administration of soluble (s)CD40L in normal rats significantly increased retinal vascular permeability and induced significant upregulation of phospho-ERK1/2, VEGF, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). sCD40L induced upregulation of VEGF, MMP-9, MCP-1 and HMGB1 in cultured Müller cells and phospo-ERK1/2, p65 subunit of NF-ĸB, VCAM-1 and VEGF in cultured HRMECS. TNF-α induced significant upregulation of CD40 in HRMECs and Müller cells and VEGF induced significant upregulation of CD40 in HRMECs. sCD40L induced proliferation and migration of HRMECs. We provide experimental evidence supporting the involvement of the CD40L-CD40 pathway and how it regulates inflammatory angiogenesis in PDR.
Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Humanos , Ratas , Animales , Retinopatía Diabética/metabolismo , Ligando de CD40/metabolismo , Células Endoteliales/metabolismo , Molécula 1 de Adhesión Celular Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Ratas Sprague-Dawley , Inflamación/metabolismo , Diabetes Mellitus/metabolismoRESUMEN
Inflammation and fibrosis are key features of proliferative vitreoretinal disorders. We aimed to define the macrophage phenotype and investigate the role of macrophage-myofibroblast transition (MMT) in the contribution to myofibroblast populations present in epiretinal membranes. Vitreous samples from proliferative diabetic retinopathy (PDR), proliferative vitreoretinopathy (PVR) and nondiabetic control patients, epiretinal fibrovascular membranes from PDR patients and fibrocellular membranes from PVR patients, human retinal Müller glial cells and human retinal microvascular endothelial cells (HRMECs) were studied by ELISA, immunohistochemistry and flow cytometry analysis. Myofibroblasts expressing α-SMA, fibroblast activation protein-α (FAP-α) and fibroblast-specific protein-1 (FSP-1) were present in all membranes. The majority of CD68+ monocytes/macrophages co-expressed the M2 macrophage marker CD206. In epiretinal membranes, cells undergoing MMT were identified by co-expression of the macrophage marker CD68 and myofibroblast markers α-SMA and FSP-1. Further analysis revealed that CD206+ M2 macrophages co-expressed α-SMA, FSP-1, FAP-α and ß-catenin. Soluble (s) CD206 and sFAP-α levels were significantly higher in vitreous samples from PDR and PVR patients than in nondiabetic control patients. The proinflammatory cytokine TNF-α and the hypoxia mimetic agent cobalt chloride induced upregulation of sFAP-α in culture media of Müller cells but not of HRMECs. The NF-Ä¸ß inhibitor BAY11-7085 significantly attenuated TNF-α-induced upregulation of sFAP-α in Müller cells. Our findings suggest that the process of MMT might contribute to myofibroblast formation in epiretinal membranes, and this transition involved macrophages with a predominant M2 phenotype. In addition, sFAP-α as a vitreous biomarker may be derived from M2 macrophages transitioned to myofibroblasts and from Müller cells.
Asunto(s)
Retinopatía Diabética , Membrana Epirretinal , Oftalmopatías , Vitreorretinopatía Proliferativa , Humanos , Células Endoteliales , Miofibroblastos , Factor de Necrosis Tumoral alfaRESUMEN
BACKGROUND: Furin converts inactive proproteins into bioactive forms. By activating proinflammatory and proangiogenic factors, furin might play a role in pathophysiology of proliferative diabetic retinopathy (PDR). METHODS: We studied vitreous samples from PDR and nondiabetic patients, epiretinal membranes from PDR patients, retinal microvascular endothelial cells (HRMECs), retinal Müller cells and rat retinas by ELISA, Western blot analysis, immunohistochemistry and immunofluorescence microscopy. We performed in vitro angiogenesis assays and assessed adherence of monocytes to HRMECs. RESULTS: Furin levels were significantly increased in PDR vitreous samples. In epiretinal membranes, immunohistochemistry analysis revealed furin expression in monocytes/macrophages, vascular endothelial cells and myofibroblasts. Furin was significantly upregulated in diabetic rat retinas. Hypoxia and TNF-α induced significant upregulation of furin in Müller cells and HRMECs. Furin induced upregulation of phospho-ERK1/2, p65 subunit of NF-κB, ADAM17 and MCP-1 in cultured Müller cells and phospho-ERK1/2 in cultured HRMECs and induced HRMECs migration. Treatment of monocytes with furin significantly increased their adhesion to HRMECs. Intravitreal administration of furin in normal rats induced significant upregulation of p65 subunit of NF-κB, phospho-ERK1/2 and ICAM-1 in the retina. Inhibition of furin with dec-CMK significantly decreased levels of MCP-1 in culture medium of Müller cells and HRMECs and significantly attenuated TNF-α-induced upregulation of p65 subunit of NF-κB, ICAM-1 and VCAM-1 in HRMECs. Dec-CMK significantly decreased adherence of monocytes to HRMECs and TNF-α-induced upregulation of adherence of monocytes to HRMECs. Treatment of HRMECs with dec-CMK significantly attenuated migration of HRMECs. CONCLUSIONS: Furin is a potential driver molecule of PDR-associated inflammation and angiogenesis.
Asunto(s)
Diabetes Mellitus Experimental , Retinopatía Diabética , Membrana Epirretinal , Furina , Animales , Diabetes Mellitus Experimental/metabolismo , Retinopatía Diabética/metabolismo , Células Endoteliales/metabolismo , Furina/metabolismo , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , FN-kappa B/metabolismo , Neovascularización Patológica/metabolismo , Proproteína Convertasas/metabolismo , Ratas , Factor de Necrosis Tumoral alfa/metabolismo , Cuerpo Vítreo/metabolismoRESUMEN
N-formyl peptide receptors (FPRs) are G protein-coupled receptors involved in the recruitment and activation of immune cells in response to pathogen-associated molecular patterns. Three FPRs have been identified in humans (FPR1-FPR3), characterized by different ligand properties, biological function and cellular distribution. Recent findings from our laboratory have shown that the peptide BOC-FLFLF (L-BOC2), related to the FPR antagonist BOC2, acts as an angiogenesis inhibitor by binding to various angiogenic growth factors, including vascular endothelial growth factor-A165 (VEGF). Here we show that the all-D-enantiomer of L-BOC2 (D-BOC2) is devoid of any VEGF antagonist activity. At variance, D-BOC2, as well as the D-FLFLF and succinimidyl (Succ)-D-FLFLF (D-Succ-F3) D-peptide variants, is endowed with a pro-angiogenic potential. In particular, the D-peptide D-Succ-F3 exerts a pro-angiogenic activity in a variety of in vitro assays on human umbilical vein endothelial cells (HUVECs) and in ex vivo and in vivo assays in chick and zebrafish embryos and adult mice. This activity is related to the capacity of D-Succ-F3 to bind FRP3 expressed by HUVECs. Indeed, the effects exerted by D-Succ-F3 on HUVECs are fully suppressed by the G protein-coupled receptor inhibitor pertussis toxin, the FPR2/FPR3 antagonist WRW4 and by an anti-FPR3 antibody. A similar inhibition was observed following WRW4-induced FPR3 desensitization in HUVECs. Finally, D-Succ-F3 prevented the binding of the anti-FPR3 antibody to the cell surface of HUVECs. In conclusion, our data demonstrate that the angiogenic activity of D-Succ-F3 is due to the engagement and activation of FPR3 expressed by endothelial cells, thus shedding a new light on the biological function of this chemoattractant receptor.
Asunto(s)
Células Endoteliales de la Vena Umbilical Humana/metabolismo , Neovascularización Fisiológica/efectos de los fármacos , Oligopéptidos/farmacología , Receptores de Formil Péptido , Humanos , Oligopéptidos/síntesis química , Oligopéptidos/química , Receptores de Formil Péptido/agonistas , Receptores de Formil Péptido/metabolismoRESUMEN
PURPOSE: To determine the expression of the endogenous anti-angiogenic and pro-fibrotic matricellular protein thrombospondin (TSP)-2 and its receptors CD36 and CD47 in proliferative diabetic retinopathy (PDR). In addition, we examined the expression of TSP-2 in the retinas of diabetic rats. METHODS: Epiretinal membranes from 14 patients with PDR and nine patients with proliferative vitreoretinopathy were studied by immunohistochemistry. Vitreous samples from 30 PDR and 25 nondiabetic patients were studied by enzyme-linked immunosorbent assay. Vitreous samples and retinas of rats were examined by Western blotting. RESULTS: In epiretinal membranes, vascular endothelial cells and myofibroblasts expressed TSP-2, CD36 and CD47. In PDR membranes, significant correlations were observed between numbers of blood vessels expressing the panendothelial cell marker CD34 and numbers of blood vessels and stromal cells expressing TSP-2, CD36 and CD47. The numbers of blood vessels and stromal cells expressing CD34, TSP-2, CD36 and CD47 were significantly higher in membranes with active neovascularization when compared with those with quiescent disease. Thrombospondin-2 levels in vitreous samples from PDR patients were significantly higher than those in control patients without diabetes (p < 0.001). Western blot analysis revealed a significant increase in the expression of intact and cleaved TSP-2 in vitreous samples from PDR patients and in the retinas of diabetic rats compared to nondiabetic controls. CONCLUSIONS: Upregulation of TSP-2 may be a protective mechanism against inflammation and angiogenesis associated with PDR.