Eun Young Choi

A novel pathway of HMGB1-mediated inflammatory cell recruitment that requires Mac-1-integrin

High‐mobility group box 1 (HMGB1) is released extracellularly upon cell necrosis acting as a mediator in tissue injury and inflammation. However, the molecular mechanisms for the proinflammatory effect of HMGB1 are poorly understood. Here, we define a novel function of HMGB1 in promoting Mac‐1‐dependent neutrophil recruitment. HMGB1 administration induced rapid neutrophil recruitment in vivo. HMGB1‐mediated recruitment was prevented in mice deficient in the β2‐integrin Mac‐1 but not in those deficient in LFA‐1. As observed by bone marrow chimera experiments, Mac‐1‐dependent neutrophil recruitment induced by HMGB1 required the presence of receptor for advanced glycation end products (RAGE) on neutrophils but not on endothelial cells. In vitro, HMGB1 enhanced the interaction between Mac‐1 and RAGE. Consistently, HMGB1 activated Mac‐1 as well as Mac‐1‐mediated adhesive and migratory functions of neutrophils in a RAGE‐dependent manner. Moreover, HMGB1‐induced activation of nuclear factor‐κB in neutrophils required both Mac‐1 and RAGE. Together, a novel HMGB1‐dependent pathway for inflammatory cell recruitment and activation that requires the functional interplay between Mac‐1 and RAGE is described here.

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.

Platelets Contribute to the Pathogenesis of Experimental Autoimmune Encephalomyelitis

Rationale: Multiple sclerosis (MS) and its mouse model, experimental autoimmune encephalomyelitis (EAE), are inflammatory disorders of the central nervous system (CNS). The function of platelets in inflammatory and autoimmune pathologies is thus far poorly defined. Objective: We addressed the role of platelets in mediating CNS inflammation in EAE. Methods and Results: We found that platelets were present in human MS lesions as well as in the CNS of mice subjected to EAE but not in the CNS from control nondiseased mice. Platelet depletion at the effector-inflammatory phase of EAE in mice resulted in significantly ameliorated disease development and progression. EAE suppression on platelet depletion was associated with reduced recruitment of leukocytes to the inflamed CNS, as assessed by intravital microscopy, and with a blunted inflammatory response. The platelet-specific receptor glycoprotein Ib&agr; (GPIb&agr;) promotes both platelet adhesion and inflammatory actions of platelets and targeting of GPIb&agr; attenuated EAE in mice. Moreover, targeting another platelet adhesion receptor, glycoprotein IIb/IIIa (GPIIb/IIIa), also reduced EAE severity in mice. Conclusions: Platelets contribute to the pathogenesis of EAE by promoting CNS inflammation. Targeting platelets may therefore represent an important new therapeutic approach for MS treatment.

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.

A Novel Function of Junctional Adhesion Molecule-C in Mediating Melanoma Cell Metastasis

Hematogenous dissemination of melanoma is a life-threatening complication of this malignant tumor. Here, we identified junctional adhesion molecule-C (JAM-C) as a novel player in melanoma metastasis to the lung. JAM-C expression was identified in human and murine melanoma cell lines, in human malignant melanoma, as well as in metastatic melanoma including melanoma lung metastasis. JAM-C expressed on both murine B16 melanoma cells as well as on endothelial cells promoted the transendothelial migration of the melanoma cells. We generated mice with inactivation of JAM-C. JAM-C(-/-) mice as well as endothelial-specific JAM-C-deficient mice displayed significantly decreased B16 melanoma cell metastasis to the lung, whereas treatment of mice with soluble JAM-C prevented melanoma lung metastasis. Together, JAM-C represents a novel therapeutic target for melanoma metastasis.

Regulation of LFA-1-dependent inflammatory cell recruitment by Cbl-b and 14-3-3 proteins

Inside-out signaling regulation of the beta2-integrin leukocyte function-associated antigen-1 (LFA-1) by different cytoplasmic proteins, including 14-3-3 proteins, is essential for adhesion and migration of immune cells. Here, we identify a new pathway for the regulation of LFA-1 activity by Cbl-b, an adapter molecule and ubiquitin ligase that modulates several signaling pathways. Cbl-b-/- mice displayed increased macrophage recruitment in thioglycollate-induced peritonitis, which was attributed to Cbl-b deficiency in macrophages, as assessed by bone marrow chimera experiments. In vitro, Cbl-b-/- bone marrow-derived mononuclear phagocytes (BMDMs) displayed increased adhesion to endothelial cells. Activation of LFA-1 in Cbl-b-deficient cells was responsible for their increased endothelial adhesion in vitro and peritoneal recruitment in vivo, as the phenotype of Cbl-b deficiency was reversed in Cbl-b-/-LFA-1-/- mice. Consistently, LFA-1-mediated adhesion of BMDM to ICAM-1 but not VLA-4-mediated adhesion to VCAM-1 was enhanced by Cbl-b deficiency. Cbl-b deficiency resulted in increased phosphorylation of T758 in the beta2-chain of LFA-1 and thereby in enhanced association of 14-3-3beta protein with the beta2-chain, leading to activation of LFA-1. Consistently, disruption of the 14-3-3/beta2-integrin interaction abrogated the enhanced ICAM-1 adhesion of Cbl-b-/- BMDMs. In conclusion, Cbl-b deficiency activates LFA-1 and LFA-1-mediated inflammatory cell recruitment by stimulating the interaction between the LFA-1 beta-chain and 14-3-3 proteins.

Inhibition of Leukocyte Adhesion by Developmental Endothelial Locus-1 (Del-1)

The leukocyte adhesion to endothelium is pivotal in leukocyte recruitment which takes place during inflammatory, autoimmune and infectious conditions. The interaction between leukocytes and endothelium requires an array of adhesion molecules expressed on leukocytes and endothelial cells, thereby promoting leukocyte recruitment into sites of inflammation and tissue injury. Intervention with the adhesion molecules provides a platform for development of anti-inflammatory therapeutics. This review will focus on developmental endothelial locus-1 (Del-1), an endogenous inhibitor of leukocyte adhesion.

Cerebrospinal Fluid-Directed rAAV9-rsATP7A Plus Subcutaneous Copper Histidinate Advance Survival and Outcomes in a Menkes Disease Mouse Model

Menkes disease is a lethal neurodegenerative disorder of copper metabolism caused by mutations in an evolutionarily conserved copper transporter, ATP7A. Based on our prior clinical and animal studies, we seek to develop a therapeutic approach suitable for application in affected human subjects, using the mottled-brindled (mo-br) mouse model that closely mimics the Menkes disease biochemical and clinical phenotypes. Here, we evaluate the efficacy of low-, intermediate-, and high-dose recombinant adeno-associated virus serotype 9 (rAAV9)-ATP7A delivered to the cerebrospinal fluid (CSF), in combination with subcutaneous administration of clinical-grade copper histidinate (sc CuHis, IND #34,166). Mutant mice that received high-dose (1.6 × 1010 vg) cerebrospinal fluid-directed rAAV9-rsATP7A plus sc copper histidinate showed 53.3% long-term (≥300-day) survival compared to 0% without treatment or with either treatment alone. The high-dose rAAV9-rsATP7A plus sc copper histidinate-treated mutant mice showed increased brain copper levels, normalized brain neurochemical levels, improvement of brain mitochondrial abnormalities, and normal growth and neurobehavioral outcomes. This synergistic treatment effect represents the most successful rescue to date of the mo-br mouse model. Based on these findings, and the absence of a large animal model, we propose cerebrospinal fluid-directed rAAV9-rsATP7A gene therapy plus subcutaneous copper histidinate as a potential therapeutic approach to cure or ameliorate Menkes disease.

615. Choroid Plexus-Targeted Viral Gene Therapy for Lysosomal Storage Diseases

Lysosomal storage diseases (LSDs) represent a category of approximately 60 inherited neurometabolic illnesses with considerable morbidity and mortality in children and adults. The burden of LSD is high due to the chronic progressive decline in the neurocognitive function of affected individuals that profoundly limits their societal integration. Current therapy for LSDs has focused on recombinant enzyme replacement therapy (ERT), which while promising, is not ideal for numerous reasons, including the short half-lives of ERT with weekly or monthly administration, potential immune responses against ERT may also reduce the effectiveness for the treatment and inefficient passage through the blood-brain barrier. The choroid plexuses are vascularized structures that project into the cerebrospinal fluid (CSF) and feature specialized polarized epithelia derived from neuroectoderm that are post-mitotic, i.e., do not undergo turnover, and produce CSF by transporting water and ions into the brain ventricles. We hypothesized that remodeling these epithelia to secrete missing lysosomal enzymes by one-time administration of a recombinant AAV vector with selective tropism for choroid plexus (e.g., serotype 5) would be an attractive strategy for long-term treatment of LSDs. Potentially this approach would result in steady secretion of the missing enzyme into the CSF, which normally carries molecules throughout the ventricular system into the subarachnoid space, and ultimately deliver enzyme to the entire brain. The cross-correction phenomenon in many LSDs would provide a further advantage. To evaluate this hypothesis in preclinical animal models, we chose two prototypical LSDs, α-mannosidosis and mucopolysaccharidosis type IIIB (MPS IIIB or Sanfilippo B syndrome). We cloned the respective cDNAs (human (h) LAMAN and NAGLU) into rAAV shuttle plasmids and generated high titer of rAAV5 expressing the enzymes. We administered viral particles to the lateral cerebroventricles sof homozygous mutant mice on day 2 or 3 of life at doses of 5 × 109 or 5 × 1010 vector genomes. In the LAMAN deficient mice, we documented dose-dependent transduction and hLAMAN mRNA expression confined to the choroid plexuses of rAAV5-treated animals. Brain biochemical analyses at 1, 2 and 6 months post-treatment documented sustained, highly statistically significant increases of LAMAN enzyme activity in the brain globally (olfactory bulb, cerebral cortex, cerebellum, brainstem). By 8 months of age, untreated mutant mice showed prominent lysosomal vacuoles in hippocampal neurons, in contrast to rAAV5-hLAMAN treated mutants for which brain histopathology was comparable to wild-type. Lysosomal associated membrane protein 1 (Lamp1) levels were normalized in AAV5-hLAMAN treated mutant brain. In MPS IIIB mutant mice, levels of NAGLU enzyme activity were 2-8 fold higher in brain sections six weeks after rAAV5-hNAGLU treatment compared to normal controls. β-Hexosaminidase activity, which is elevated in MPS IIIB, was reduced to heterozygote carrier levels. Tissue evaluations by immunohistochemistry showed robust hNAGLU expression in the choroid plexus epithelia. Lamp1 expression was significantly reduced in hippocampus and frontal cortex of treated mice. The approach outlined herein challenges existing treatment modalities for LSDs, by exploiting the ability of choroid plexus-targeted gene therapy to restore missing or defective lysosomal enzymes at concentrations and distributions in CSF suitable for disease correction. The potential impact on clinical practice in the field of LSD is high since, if these results extend to larger animal disease models, nonhuman primates, and human subjects, the largest current barriers to health for affected patients would be circumvented.