Matina Economopoulou

Del-1, an Endogenous Leukocyte-Endothelial Adhesion Inhibitor, Limits Inflammatory Cell Recruitment

Leukocyte recruitment to sites of infection or inflammation requires multiple adhesive events. Although numerous players promoting leukocyte-endothelial interactions have been characterized, functionally important endogenous inhibitors of leukocyte adhesion have not been identified. Here we describe the endothelially derived secreted molecule Del-1 (developmental endothelial locus–1) as an anti-adhesive factor that interferes with the integrin LFA-1–dependent leukocyte-endothelial adhesion. Endothelial Del-1 deficiency increased LFA-1–dependent leukocyte adhesion in vitro and in vivo. Del-1–/– mice displayed significantly higher neutrophil accumulation in lipopolysaccharide-induced lung inflammation in vivo, which was reversed in Del-1/LFA-1 double-deficient mice. Thus, Del-1 is an endogenous inhibitor of inflammatory cell recruitment and could provide a basis for targeting leukocyte-endothelial interactions in disease.

Complement-mediated inhibition of neovascularization reveals a point of convergence between innate immunity and angiogenesis

Beyond its role in immunity, complement mediates a wide range of functions in the context of morphogenetic or tissue remodeling processes. Angiogenesis is crucial during tissue remodeling in multiple pathologies; however, the knowledge about the regulation of neovascularization by the complement components is scarce. Here we studied the involvement of complement in pathological angiogenesis. Strikingly, we found that mice deficient in the central complement component C3 displayed increased neovascularization in the model of retinopathy of prematurity (ROP) and in the in vivo Matrigel plug assay. In addition, antibody-mediated blockade of C5, treatment with C5aR antagonist, or C5aR deficiency in mice resulted in enhanced pathological retina angiogenesis. While complement did not directly affect angiogenesis-related endothelial cell functions, we found that macrophages mediated the antiangiogenic activity of complement. In particular, C5a-stimulated macrophages were polarized toward an angiogenesis-inhibitory phenotype, including the up-regulated secretion of the antiangiogenic soluble vascular endothelial growth factor receptor-1. Consistently, macrophage depletion in vivo reversed the increased neovascularization associated with C3- or C5aR deficiency. Taken together, complement and in particular the C5a-C5aR axes are potent inhibitors of angiogenesis.

Lipoprotein(a) in atherosclerotic plaques recruits inflammatory cells through interaction with Mac-1 integrin

Lipoprotein(a) [Lp(a)], consisting of LDL and the unique constituent apolipoprotein(a) [apo(a)], which contains multiple repeats resembling plasminogen kringle 4, is considered a risk factor for the development of atherosclerotic disorders. However, the underlying mechanisms for the atherogenicity of Lp(a) are not completely understood. Here, we define a novel function of Lp(a) in promoting inflammatory cell recruitment that may contribute to its atherogenicity. Through its apo(a) moiety Lp(a) specifically interacts with the ?2‐integrin Mac‐1, thereby promoting the adhesion of monocytes and their transendothelial migration in a Mac‐1‐dependent manner. Interestingly, the interaction between Mac‐1 and Lp(a) was strengthened in the presence of proatherogenic homocysteine and was blocked by plasminogen/angiostatin kringle 4. Through its interaction with Mac‐1, Lp(a) induced activation of the proinflammatory transcription factor NF?B, as well as the NF?B‐related expression of prothrombotic tissue factor. In atherosclerotic coronary arteries Lp(a) was found to be localized in close proximity to Mac‐1 on infiltrating mononuclear cells. Taken together, our data demonstrate that Lp(a), via its apo(a) moiety, is a ligand for the ?2‐integrin Mac‐1, thereby facilitating inflammatory cell recruitment to atherosclerotic plaques. These observations suggest a novel mechanism for the atherogenic properties of Lp(a).

Histone H2AX is integral to hypoxia-driven neovascularization

H2A histone family member X (H2AX, encoded by H2AFX) and its C-terminal phosphorylation (γ-H2AX) participates in the DNA damage response and mediates DNA repair. Hypoxia is a physiological stress that induces a replication-associated DNA damage response. Moreover, hypoxia is the major driving force for neovascularization, as the hypoxia-mediated induction of vascular growth factors triggers endothelial cell proliferation. Here we studied the role of the hypoxia-induced DNA damage response in endothelial cell function and in hypoxia-driven neovascularization in vivo. Hypoxia induced replication-associated generation of γ-H2AX in endothelial cells in vitro and in mice. Both in cultured cells and in mice, endothelial cell proliferation under hypoxic conditions was reduced by H2AX deficiency. Whereas developmental angiogenesis was not affected in H2afx−/− mice, hypoxia-induced neovascularization during pathologic proliferative retinopathy, in response to hind limb ischemia or during tumor angiogenesis was substantially lower in H2afx−/− mice. Moreover, endothelial-specific H2afx deletion resulted in reduced hypoxia-driven retina neovascularization and tumor neovascularization. Our findings establish that H2AX, and hence activation of the DNA repair response, is needed for endothelial cells to maintain their proliferation under hypoxic conditions and is crucial for hypoxia-driven neovascularization.

EphrinB reverse signaling contributes to endothelial and mural cell assembly into vascular structures

EphrinB transmembrane ligands and their cognate EphB receptor tyrosine kinases regulate vascular development through bidirectional cell-to-cell signaling, but little is known about the role of EphrinB during postnatal vascular remodeling. We report that EphrinB is a critical mediator of postnatal pericyte-to-endothelial cell assembly into vascular structures. This function is dependent upon extracellular matrix-supported cell-to-cell contact, engagement of EphrinB by EphB receptors expressed on another cell, and Src-dependent phosphorylation of the intracytoplasmic domain of EphrinB. Phosphorylated EphrinB marks angiogenic blood vessels in the developing and hypoxic retina, the wounded skin, and tumor tissue, and is detected at contact points between endothelial cells and pericytes. Furthermore, inhibition ofEphrinB activity prevents proper assembly of pericytes and endothelial cells into vascular structures. These results reveal a role for EphrinB signaling in orchestrating pericyte/endothelial cell assembly, and suggest that therapeutic targeting of EphrinB may prove useful for disrupting angiogenesis when it contributes to disease.

The Complement Anaphylatoxin C5a Receptor Contributes to Obese Adipose Tissue Inflammation and Insulin Resistance

Obese adipose tissue (AT) inflammation contributes critically to development of insulin resistance. The complement anaphylatoxin C5a receptor (C5aR) has been implicated in inflammatory processes and as regulator of macrophage activation and polarization. However, the role of C5aR in obesity and AT inflammation has not been addressed. We engaged the model of diet-induced obesity and found that expression of C5aR was significantly upregulated in the obese AT, compared with lean AT. In addition, C5a was present in obese AT in the proximity of macrophage-rich crownlike structures. C5aR-sufficient and -deficient mice were fed a high-fat diet (HFD) or a normal diet (ND). C5aR deficiency was associated with increased AT weight upon ND feeding in males, but not in females, and with increased adipocyte size upon ND and HFD conditions in males. However, obese C5aR−/− mice displayed improved systemic and AT insulin sensitivity. Improved AT insulin sensitivity in C5aR−/− mice was associated with reduced accumulation of total and proinflammatory M1 macrophages in the obese AT, increased expression of IL-10, and decreased AT fibrosis. In contrast, no difference in β cell mass was observed owing to C5aR deficiency under an HFD. These results suggest that C5aR contributes to macrophage accumulation and M1 polarization in the obese AT and thereby to AT dysfunction and development of AT insulin resistance.

Expression, localization, and function of junctional adhesion molecule-C (JAM-C) in human retinal pigment epithelium.

PURPOSE To determine the localization of JAM-C in human RPE and characterize its functions. METHODS Immunofluorescence, Western blot, and PCR was used to identify the localization and expression of JAM-C, ZO-1, N-cadherin, and ezrin in cultures of human fetal RPE (hfRPE) with or without si-RNA mediated JAM-C knockdown and in adult native RPE wholemounts. A transepithelial migration assay was used to study the migration of leukocytes through the hfRPE monolayer. RESULTS JAM-C localized at the tight junctions of cultured hfRPE cells and adult native RPE. During initial junction formation JAM-C was recruited to the primordial cell-cell contacts and after JAM-C knockdown, the organization of N-cadherin and ZO-1 at those contacts was disrupted. JAM-C knockdown caused a delay in the hfRPE cell polarization, as shown by reduced apical staining of ezrin. JAM-C inhibition significantly decreased the chemokine-induced transmigration of granulocytes but not monocytes through the hfRPE monolayer. CONCLUSIONS JAM-C localizes specifically in the tight junctions of hfRPE and adult native RPE. It is important for tight junction formation in hfRPE, possibly by regulating the recruitment of N-cadherin and ZO-1 at the cell-cell contacts, and has a role in the polarization of hfRPE cells. Finally, JAM-C promotes the basal-to-apical transmigration of granulocytes but not monocytes through the hfRPE monolayer.

Opposing effects of HIF1α and HIF2α on chromaffin cell phenotypic features and tumor cell proliferation: Insights from MYC-associated factor X

Pheochromocytomas and paragangliomas (PPGLs) are catecholamine‐producing chromaffin cell tumors with diverse phenotypic features reflecting mutations in numerous genes, including MYC‐associated factor X (MAX). To explore whether phenotypic differences among PPGLs reflect a MAX‐mediated mechanism and opposing influences of hypoxia‐inducible factor (HIF)s HIF2α and HIF1α, we combined observational investigations in PPGLs and gene‐manipulation studies in two pheochromocytoma cell lines. Among PPGLs from 140 patients, tumors due to MAX mutations were characterized by gene expression profiles and intermediate phenotypic features that distinguished these tumors from other PPGLs, all of which fell into two expression clusters: one cluster with low expression of HIF2α and mature phenotypic features and the other with high expression of HIF2α and immature phenotypic features due to mutations stabilizing HIFs. Max‐mutated tumors distributed to a distinct subcluster of the former group. In cell lines lacking Max, re‐expression of the gene resulted in maturation of phenotypic features and decreased cell cycle progression. In cell lines lacking Hif2α, overexpression of the gene led to immature phenotypic features, failure of dexamethasone to induce differentiation and increased proliferation. HIF1α had opposing actions to HIF2α in both cell lines, supporting evolving evidence of their differential actions on tumorigenic processes via a MYC/MAX‐related pathway. Requirement of a fully functional MYC/MAX complex to facilitate differentiation explains the intermediate phenotypic features in tumors due to MAX mutations. Overexpression of HIF2α in chromaffin cell tumors due to mutations affecting HIF stabilization explains their proliferative features and why the tumors fail to differentiate even when exposed locally to adrenal steroids.

Developmental endothelial locus-1 is a homeostatic factor in the central nervous system limiting neuroinflammation and demyelination.

Inflammation in the central nervous system (CNS) and disruption of its immune privilege are major contributors to the pathogenesis of multiple sclerosis (MS) and of its rodent counterpart, experimental autoimmune encephalomyelitis (EAE). We have previously identified developmental endothelial locus-1 (Del-1) as an endogenous anti-inflammatory factor, which inhibits integrin-dependent leukocyte adhesion. Here we show that Del-1 contributes to the immune privilege status of the CNS. Intriguingly, Del-1 expression decreased in chronic-active MS lesions and in the inflamed CNS in the course of EAE. Del-1-deficiency was associated with increased EAE severity, accompanied by increased demyelination and axonal loss. As compared with control mice, Del-1−/− mice displayed enhanced disruption of the blood–brain barrier and increased infiltration of neutrophil granulocytes in the spinal cord in the course of EAE, accompanied by elevated levels of inflammatory cytokines, including interleukin-17 (IL-17). The augmented levels of IL-17 in Del-1-deficiency derived predominantly from infiltrated CD8+ T cells. Increased EAE severity and neutrophil infiltration because of Del-1-deficiency was reversed in mice lacking both Del-1 and IL-17 receptor, indicating a crucial role for the IL-17/neutrophil inflammatory axis in EAE pathogenesis in Del-1−/− mice. Strikingly, systemic administration of Del-1-Fc ameliorated clinical relapse in relapsing–remitting EAE. Therefore, Del-1 is an endogenous homeostatic factor in the CNS protecting from neuroinflammation and demyelination. Our findings provide mechanistic underpinnings for the previous implication of Del-1 as a candidate MS susceptibility gene and suggest that Del-1-centered therapeutic approaches may be beneficial in neuroinflammatory and demyelinating disorders.

Adipocyte-Specific Hypoxia-Inducible Factor 2α Deficiency Exacerbates Obesity-Induced Brown Adipose Tissue Dysfunction and Metabolic Dysregulation

ABSTRACT Angiogenesis is a central regulator for white (WAT) and brown (BAT) adipose tissue adaptation in the course of obesity. Here we show that deletion of hypoxia-inducible factor 2α (HIF2α) in adipocytes (by using Fabp4-Cre transgenic mice) but not in myeloid or endothelial cells negatively impacted WAT angiogenesis and promoted WAT inflammation, WAT dysfunction, hepatosteatosis, and systemic insulin resistance in obesity. Importantly, adipocyte HIF2α regulated vascular endothelial growth factor (VEGF) expression and angiogenesis of obese BAT as well as its thermogenic function. Consistently, obese adipocyte-specific HIF2α-deficient mice displayed BAT dysregulation, associated with reduced levels of uncoupling protein 1 (UCP1) and a dysfunctional thermogenic response to cold exposure. VEGF administration reversed WAT and BAT inflammation and BAT dysfunction in adipocyte HIF2α-deficient mice. Together, our findings show that adipocyte HIF2α is protective against maladaptation to obesity and metabolic dysregulation by promoting angiogenesis in both WAT and BAT and by counteracting obesity-mediated BAT dysfunction.