Angiogenic endothelium shows lactadherin-dependent phagocytosis of aged erythrocytes and apoptotic cells

Abstract

IntroductionCells that are going into apoptosis expose phosphatidylserine (PS) on their surface that serves as an "eat me" signal for both professional phagocytes (such as macrophages, B lymphocytes, and immature dendritic cells) and nonprofessional phagocytes (such as fibroblasts and epithelial cells). 1 Phagocytes recognize apoptotic cells directly via the PS receptor or indirectly via ␣ v -integrins that are present on the phagocyte surface. 2 In the latter process, ␣ v ␤ 3 and ␣ v ␤ 5 integrins recognize PS through an opsonin called lactadherin, also known as milk fat globule epidermal growth factor-8 (MFG-E8). 3,4 This opsonin contains a PS-binding C-domain and an RGD-motif present in the epidermal growth factor domain, which binds to ␣ v ␤ 3 and ␣ v ␤ 5 integrins. 2,[5][6][7] Rapid clearance of apoptotic cells prevents the release of potentially harmful or immunogenic intracellular materials from these cells. 8 Lactadherin-deficient mice have shown impaired phagocytosis of apoptotic cells, induction of autoimmune diseases, and autoantibody production. 9,10 ␣ v -Integrins are densely present on macrophages and dendritic cells and play an important role in phagocytosis. 11 Notably, these integrins are also overexpressed on angiogenic endothelium 12 where they play an important role in cell adhesion and migration. 13,14 Recently, Silvestre et al discovered that lactadherin is expressed in and around (angiogenic) blood vessels in an ischemic hind limb model and that the protein seems to play a crucial role in vascular endothelial growth factor (VEGF)-dependent neovascularization. 15 As angiogenesis is also a hallmark of tumor growth and tumor angiogenic endothelium expresses ␣ v -integrins, in this study we have explored the possible role of lactadherin-induced phagocytosis by angiogenic tumor endothelial cells. Methods Cell linesB16.F10 murine melanoma cells (ATCC, Manassas, VA) were cultured on regular FCS-enriched DMEM. Human umbilical vein endothelial cells (HUVECs) were cultured on EGM-2 endothelial cell growth medium-2 (Cambrex, East Rutherford, NJ) consisting of EBM-2 medium supplemented with an EGM-2 bullet kit (containing growth factors, 2% FBS, and antibiotics). HUVECs were used as an ␣ v ␤ 3 -expressing model for proliferating endothelium to a maximal passage number of 8. Lactadherin purificationBovine lactadherin was purified as previously described. 16 Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and N-terminal amino acid sequencing and shown to be Submitted June 8, 2007; accepted February 13, 2008. Prepublished online as Blood First Edition paper, February 21, 2008 DOI 10.1182 DOI 10. /blood-2007 The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked ''advertisement'' in accordance with 18 USC section 1734. For personal use only. on May 12, 2018. by guest www.bloodjournal.org From 97% to...

A role for activated endothelial cells in red blood cell clearance: implications for vasopathology

“…Previously, we showed that endothelial cells are capable of lactadherin-dependent phagocytosis of RGD-modified RBC. 16,20 In the present study we provide evidence for endothelial cell erythrophagocytosis via this pathway by reporting on the interactions between PS-exposing RBC and endothelium in in vitro models mimicking (patho)physiological conditions. We show that, in the presence of lactadherin, PSexposing RBC have severely reduced deformability, and are phagocytosed by endothelial cells under both static and flow conditions.…”

“…Clearance and subsequent processing of innate cells should initiate an immunosuppressive effect rather than an immunostimulatory effect, to prevent autoimmune reactions and inflammation. 19 Since endothelial cells, like macrophages, are equipped with all elements of the immunosuppressive PS-lactadherin αv-integrin pathway 16,20 it seems reasonable to assume that under certain circumstances they actively engage this pathway. Previously, we showed that endothelial cells are capable of lactadherin-dependent phagocytosis of RGD-modified RBC.…”

“…13,14 Lactadherin is expressed by subsets of macrophages, 15 and in and around blood vessels. 16,17 The second EGF-repeat of lactadherin contains an Arg-Gly-Asp (RGD) motif that binds to αvβ3 and αvβ5 integrin expressed on macrophages, epithelial cells, and vascular cells. 12,13,18 The integrin expression density is dependent on cellular origin and state of activation.…”

Milk fat globule—epidermal growth factor—factor VIII (MFGE8)/lactadherin promotes bladder tumor development

“…MFGE8 displays three types of functions. First, MFGE8 promotes phagocytosis of apoptotic cells by avb3/b5 integrin-expressing phagocytes (Hanayama et al, 2002), endothelial and epithelial cells (Nandrot et al, 2007;Fens et al, 2008). In Mfge8-deficient mice, impaired elimination of apoptotic cells leads to autoimmune symptoms in old animals (Hanayama et al, 2004), and mammary gland inflammation after repeated pregnancies in females (Atabai et al, 2005;Hanayama and Nagata, 2005).…”

Human endometrial milk fat globule-epidermal growth factor 8 (MFGE8) is up regulated by estradiol at the transcriptional level, and its secretion via microvesicles is stimulated by human chorionic gonadotropin (hCG)

“…In the breast it promotes phagocytosis of apoptotic cells by ανβ3/β5 integrin-expressing phagocytes [20], as well as in endothelial and epithelial cells [21,22]. Prolactin is a potent inducer of MFGE8 expression in macrophages [23].…”