Synergistic Actions of Blocking Angiopoietin-2 and Tumor Necrosis Factor-α in Suppressing Remodeling of Blood Vessels and Lymphatics in Airway Inflammation

Remodeling of blood vessels and lymphatics are prominent features of sustained inflammation. Angiopoietin-2 (Ang2)/Tie2 receptor signaling and tumor necrosis factor-α (TNF)/TNF receptor signaling are known to contribute to these changes in airway inflammation after Mycoplasma pulmonis infection in mice. We determined whether Ang2 and TNF are both essential for the remodeling on blood vessels and lymphatics, and thereby influence the actions of one another. Their respective contributions to the initial stage of vascular remodeling and sprouting lymphangiogenesis were examined by comparing the effects of function-blocking antibodies to Ang2 or TNF, given individually or together during the first week after infection. As indices of efficacy, vascular enlargement, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic vessel sprouting were assessed. Inhibition of Ang2 or TNF alone reduced the remodeling of blood vessels and lymphatics, but inhibition of both together completely prevented these changes. Genome-wide analysis of changes in gene expression revealed synergistic actions of the antibody combination over a broad range of genes and signaling pathways involved in inflammatory responses. These findings demonstrate that Ang2 and TNF are essential and synergistic drivers of remodeling of blood vessels and lymphatics during the initial stage of inflammation after infection. Inhibition of Ang2 and TNF together results in widespread suppression of the inflammatory response.Remodeling of blood vessels and lymphatics contributes to the pathophysiology of many chronic inflammatory diseases, including asthma, chronic bronchitis, chronic obstructive pulmonary disease, inflammatory bowel disease, and psoriasis.^{1, 2, 3} When inflammation is sustained, capillaries acquire venule-like properties that expand the sites of plasma leakage and leukocyte influx. Consistent with this transformation, the remodeled blood vessels express P-selectin, intercellular adhesion molecule 1 (ICAM-1), EphB4, and other venular markers.^{4, 5, 6} The changes are accompanied by remodeling of pericytes and disruption of pericyte-endothelial crosstalk involved in blood vessel quiescence.⁷ Remodeling of blood vessels is accompanied by plasma leakage, inflammatory cell influx, and sprouting lymphangiogenesis.^{6, 8, 9}Mycoplasma pulmonis infection causes sustained inflammation of the respiratory tract of rodents.¹⁰ This infection has proved useful for dissecting the features and mechanisms of vascular remodeling and lymphangiogenesis.^{6, 9, 10} At 7 days after infection, there is widespread conversion of capillaries into venules, pericyte remodeling, inflammatory cell influx, and lymphatic vessel sprouting in the airways and lung.^{4, 5, 6, 7, 8, 9} Many features of chronic M. pulmonis infection in mice are similar to Mycoplasma pneumoniae infection in humans.¹¹Angiopoietin-2 (Ang2) is a context-dependent antagonist of Tie2 receptors^{12, 13} that is important for prenatal and postnatal remodeling of blood vessels and lymphatic vessels.^{13, 14, 15} Ang2 promotes vascular remodeling,^{4, 5} lymphangiogenesis,^{15, 16, 17} and pericyte loss¹⁸ in disease models in mice. Mice genetically lacking Ang2 have less angiogenesis, lymphangiogenesis, and neutrophil recruitment in inflammatory bowel disease.³ Ang2 has proved useful as a plasma biomarker of endothelial cell activation in acute lung injury, sepsis, hypoxia, and cancer.¹⁹Like Ang2, tumor necrosis factor (TNF)-α is a mediator of remodeling of blood vessels and lymphatics.^{8, 9, 20, 21} TNF triggers many components of the inflammatory response, including up-regulation of expression of vascular cell adhesion molecule-1, ICAM-1, and other endothelial cell adhesion molecules.²² TNF inhibitors reduce inflammation in mouse models of inflammatory disease^{23, 24} and are used clinically in the treatment of rheumatoid arthritis, ankylosing spondylitis, Crohn''s disease, psoriatic arthritis, and some other inflammatory conditions.^{24, 25} Indicative of the complex role of TNF in disease, inhibition or deletion of TNF can increase the risk of serious infection by bacterial, mycobacterial, fungal, viral, and other opportunistic pathogens.²⁶TNF and Ang2 interact in inflammatory responses. TNF increases Ang2 expression in endothelial cells in a time- and dose-dependent manner, both in blood vessels²⁷ and lymphatics.¹⁶ Administration of TNF with Ang2 increases cell adhesion molecule expression more than TNF alone.^{16, 28} Similarly, Ang2 can promote corneal angiogenesis in the presence of TNF, but not alone.²⁹ In mice that lack Ang2, TNF induces leukocyte rolling but not adherence to the endothelium.²⁸ Ang2 also augments TNF production by macrophages.^{30, 31} Inhibition of Ang2 and TNF together with a bispecific antibody can ameliorate rheumatoid arthritis in a mouse model.³²With this background, we sought to determine whether Ang2 and TNF act together to drive the remodeling of blood vessels and lymphatics in the initial inflammatory response to M. pulmonis infection. In particular, we asked whether Ang2 and TNF have synergistic actions in this setting. The approach was to compare the effects of selective inhibition of Ang2 or TNF, individually or together, and then assess the severity of vascular remodeling, endothelial leakiness, venular marker expression, pericyte changes, and lymphatic sprouting. Functional consequences of genome-wide changes in gene expression were analyzed by Ingenuity Pathway Analysis (IPA)^{33, 34} and the Database for Annotation, Visualization and Integrated Discovery (DAVID).³⁵ The studies revealed that inhibition of Ang2 and TNF together, but not individually, completely prevented the development of vascular remodeling and lymphatic sprouting and had synergistic effects in suppressing gene expression and cellular pathways activated during the initial stage of the inflammatory response.     

Enoxaparin Improves the Course of Dextran Sodium Sulfate-Induced Colitis in Syndecan-1-Deficient Mice

Syndecan-1 (Sdc1) plays a major role in wound healing and modulates inflammatory responses. Sdc1 expression is reduced in lesions of patients with ulcerative colitis. The aim of this study was to investigate the role of Sdc1 in murine dextran sodium sulfate (DSS)-induced colitis. DSS colitis was induced in Sdc1-deficient (knockout (KO)) and wild-type mice by oral administration of 3% DSS. KO mice exhibited a significantly increased lethality as compared with wild-type controls (61 versus 5%, P < 0.05). Impaired mucosal healing and prolonged recruitment of inflammatory cells in KO mice were accompanied by significant up-regulation of tumor necrosis factor-α, CC chemokine ligand 3/macrophage inflammatory protein-1α, and vascular cell adhesion molecule-1, as determined by histological correlation between 0 and 15 days after colitis induction, TaqMan low-density array analysis, and quantitative real-time PCR. Treatment from days 7 through 14 with enoxaparin, a functional analogue of the Sdc1 heparan sulfate chains, significantly reduced lethality of KO mice due to DSS-induced colitis, which was correlated with improved mucosal healing. In vitro, Sdc1-deficient polymorphonuclear cells displayed increased adhesion to endothelial cells and intercellular adhesion molecule-1, and enoxaparin reverted adhesion to wild-type levels. Small interfering RNA-mediated knockdown of Sdc1 expression resulted in reduced basic fibroblast growth factor-mediated mitogen-activated protein kinase signaling and reduced Caco-2 cell proliferation. We conclude that Sdc1 has a protective effect during experimental colitis. The modification of missing Sdc1 function by heparin analogues may emerge as a promising anti-inflammatory approach.Syndecan-1 (Sdc1) is the most important representative of the heparan sulfate proteoglycans (HSPGs) covering epithelial cell surfaces.¹ It serves multiple biological roles, such as cell-matrix interactions, modulation of inflammatory responses, tumorigenesis, and wound healing.^2–4 The highly conserved cytoplasmic domains of Sdc1 interact with scaffolding proteins and participate in integrin-mediated signaling events, thus providing a physical and functional link to the cytoskeleton. In addition, most of the extracellular-binding interactions are mediated by the heparan sulfate chains, which are structurally and functionally related to heparin, an extensively sulfated and epimerized derivative of heparan sulfate.¹ Sdc1 serves as a coreceptor for several tyrosine kinase receptors. For example, it increases the activity of the complex of basic fibroblast growth factor (bFGF) and the FGF receptor and, therefore, contributes to improved wound healing via stimulation of keratinocyte proliferation.^1,5 A role for Sdc1 in wound repair in vivo has been demonstrated in Sdc1-deficient (Sdc1-knockout (KO)) mice, which show delayed skin and corneal wound healing⁵ and functionally adverse repair following experimental myocardial infarction due to dysregulation of chemokine expression and matrix metalloproteinase-mediated tissue remodeling.⁶ Sdc1 forms chemotactic gradients due to binding of chemokines on heparan sulfate chains of the molecule. Therefore, Sdc1 is able to act as coreceptor for chemokine signaling.^7,8 In addition, endothelial leukocyte recruitment and extravasation is modulated by Sdc1, possibly via interference with heparin-binding adhesion molecule function.^9–12Day et al¹³ described in 1999 a reduced expression of Sdc1 in patients with ulcerative colitis, which was linked to disrupted healing of colonic ulcers. In addition, this group demonstrated the benefit of the Sdc1 ectodomain for the FGF-induced proliferation of intestinal epithelial cell lines in vitro. The function of Sdc1 could be restored with heparin, representing a highly sulfated and epimerized form of heparan sulfate, the major functional constituent of the Sdc1 ectodomain.Heparin sees widespread use as anticoagulant drug based on its antithrombin III-activating properties. Enoxaparin, is a low molecular weight heparin with similar features in vitro and in vivo like heparin; however, it exhibits a more favorable pharmacological side effect profile. Both low molecular weight heparins (enoxaparin) as well as heparin were recently found to possess anti-inflammatory properties.¹⁴The hypothesis of Sdc1 being involved in the pathogenesis of ulcerative colitis is underlined by multiple clinical observations of patients who have been treated with heparins for different reasons.¹⁵ In a number of cases, this treatment has lead to an improved course of disease. A limited number of uncontrolled clinical trials with heparins in the treatment of low to medium active ulcerative colitis showed a variable outcome,^16–18 which may be explained by variations in treatment regimes that may have failed to include the optimal dose, class of heparin, and mode of delivery. For example, most studies have involved either i.v. or s.c. delivery of heparin, whereas a more appropriate mode of delivery for stimulating mucosal healing might be the topical application or microsphere-mediated delivery of heparin.^14,15 Furthermore, the outcome of heparin therapy may depend on the degree to which Sdc1 expression is reduced in inflammatory bowel disease (IBD) patients.¹⁹Moreover, the expression of Sdc1 and the proinflammatory cytokine tumor necrosis factor-α (TNF-α) are inversely correlated in the colonic mucosa of patients with Crohn''s disease,²⁰ and a reduction of Sdc1 expression has been shown to result in increased TNF-α signaling in an in vitro model of protein-losing enteropathy,^19,21 further suggesting a regulatory role for Sdc1 in proinflammatory cytokine signaling.In this study, our goal was to characterize the impact of a Sdc1 deficiency on the dextran sodium sulfate (DSS)-induced colitis of the mouse. Furthermore, the efficacy of low molecular weight heparin to restore altered wound healing was studied in vivo. In addition, in vitro trials were performed to study the role of Sdc1 deficiency in the adhesion and transmigration of leukocytes under inflammatory conditions.     

Serum Antibodies to N-Glycolylneuraminic Acid Are Elevated in Duchenne Muscular Dystrophy and Correlate with Increased Disease Pathology in Cmah−/−mdx Mice

Humans cannot synthesize the common mammalian sialic acid N-glycolylneuraminic acid (Neu5Gc) because of an inactivating deletion in the cytidine-5''-monophospho-(CMP)–N-acetylneuraminic acid hydroxylase (CMAH) gene responsible for its synthesis. Human Neu5Gc deficiency can lead to development of anti-Neu5Gc serum antibodies, the levels of which can be affected by Neu5Gc-containing diets and by disease. Metabolic incorporation of dietary Neu5Gc into human tissues in the face of circulating antibodies against Neu5Gc-bearing glycans is thought to exacerbate inflammation-driven diseases like cancer and atherosclerosis. Probing of sera with sialoglycan arrays indicated that patients with Duchenne muscular dystrophy (DMD) had a threefold increase in overall anti-Neu5Gc antibody titer compared with age-matched controls. These antibodies recognized a broad spectrum of Neu5Gc-containing glycans. Human-like inactivation of the Cmah gene in mice is known to modulate severity in a variety of mouse models of human disease, including the X chromosome–linked muscular dystrophy (mdx) model for DMD. Cmah^−/−mdx mice can be induced to develop anti–Neu5Gc-glycan antibodies as humans do. The presence of anti-Neu5Gc antibodies, in concert with induced Neu5Gc expression, correlated with increased severity of disease pathology in Cmah^−/−mdx mice, including increased muscle fibrosis, expression of inflammatory markers in the heart, and decreased survival. These studies suggest that patients with DMD who harbor anti-Neu5Gc serum antibodies might exacerbate disease severity when they ingest Neu5Gc-rich foods, like red meats.

Sialic acids (Sias) are negatively charged monosaccharides commonly found on the outer ends of glycan chains on glycoproteins and glycolipids in mammalian cells.¹ Although Sias are necessary for mammalian embryonic development,^1,2 they also have much structural diversity, with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) comprising the two most abundant Sia forms in most mammalian tissues. Neu5Gc differs from Neu5Ac by having an additional oxygen at the 5-N-acyl position.³ Neu5Gc synthesis requires the cytidine-5''-monophospho (CMP)-Neu5Ac hydroxylase gene, or CMAH, which encodes a hydroxylase that converts CMP-Neu5Ac to CMP-Neu5Gc.^4,5 CMP-Neu5Ac and CMP-Neu5Gc can be utilized by the >20 sialyltransferases to attach Neu5Ac or Neu5Gc, respectively, onto glycoproteins and glycolipids.^1,3Humans cannot synthesize Neu5Gc, because of an inactivating deletion in the human CMAH gene that occurred approximately 2 to 3 million years ago.⁶ This event fundamentally changed the biochemical nature of all human cell membranes, eliminating millions of oxygen atoms on Sias on the glycocalyx of almost every cell type in the body, which instead present as an excess of Neu5Ac. Consistent with the proposed timing of this mutation at around the emergence of the Homo lineage, mice with a human-like inactivation of CMAH have an enhanced ability for sustained aerobic exercise,⁷ which may have provided an evolutionary advantage. In this regard, it is also interesting that the mild phenotype of X chromosome–linked muscular dystrophy (mdx) mice with a dystrophin mutation that causes Duchenne muscular dystrophy (DMD) in humans is exacerbated and becomes more human-like on mating into a human-like CMAH null state.⁸Inactivation of CMAH in humans also fundamentally changed the immunologic profile of humans. Almost all humans consume Neu5Gc from dietary sources (particularly the red meats beef, pork, and lamb), which can be taken up by cells through a salvage pathway, sometimes allowing for Neu5Gc expression on human cell surfaces.9, 10, 11, 12, 13 Meanwhile, most humans have some level of anti–Neu5Gc-glycan antibodies, defining Neu5Gc-bearing glycans as xeno-autoantigens recognized by the immune system.13, 14, 15, 16 Humans develop antibodies to Neu5Gc not long after weaning, likely triggered by Neu5Gc incorporation into lipo-oligosaccharides of commensal bacteria in the human upper airways.¹³ The combination of xeno-autoantigens and such xeno-autoantibodies generates xenosialitis, a process that has been shown to accelerate progression of cancer and atherosclerosis in mice with a human-like CMAH deletion in the mouse Cmah gene.^17,18 Inactivation of mouse Cmah also leads to priming of macrophages and monocytes¹⁹ and enhanced reactivity²⁰ that can hyperactivate immune responses. Cmah deletion in mice also causes hearing loss via increased oxidative stress,^21,22 diabetes in obese mice,²³ relative infertility,²⁴ delayed wound healing,²¹ mitochondrial dysfunction,²² changed metabolic state,²⁵ and decreased muscle fatigability.⁷Given that Cmah deletion can hyperactivate cellular immune responses, it is perhaps not surprising that the crossing of Cmah deletion in mouse models of various human diseases, to humanize their sialic acid repertoire, can alter pathogenic disease states and disease outcomes. This is true of cancer burden from transplantation of cancer cells into mice,¹⁷ infectious burden of induced bacterial infections in mice,^13,18,19 and muscle disease burden in response to Cmah deletion in the mdx model of Duchenne muscular dystrophy⁸ and the α sarcoglycan (Sgca) deletion model of limb girdle muscular dystrophy 2D.²⁶ The mdx mice possess a mutation in the dystrophin (Dmd) gene that prevents dystrophin protein expression in almost all muscle cells,²⁷ making it a good genetic model for DMD, which also arises from lack of dystrophin protein expression.^28,29 These mdx mice, however, do not display the severe onset of muscle weakness and overall disease severity found in children with DMD, suggesting that additional genetic modifiers are at play to lessen mouse disease severity, some of which have been described.30, 31, 32, 33, 34, 35, 36 Cmah deletion worsens muscle inflammation, in particular recruitment of macrophages to muscle with concomitant increases in cytokines known to recruit them, increases complement deposition, increases muscle wasting, and premature death in a fraction of affected mdx mice.⁸ Cmah-deficient mdx mice have changed cardiac function.³⁷ Prior studies⁸ show that about half of all mice display induced antibodies to Neu5Gc, which correlates well with the number of animals showing premature death in the 6- to 12-month period. Unpublished subsequent studies suggest that Cmah^−/−mdx mice that lack xeno-autoimmunity often have less severe disease, which likely causes selection for more efficient breeders lacking Neu5Gc immunity over time. Current studies were designed to re-introduce Neu5Gc xeno-autoimmunity into serum-naive Cmah^−/−mdx mice and describe the impact of xenosialitis on disease pathogenesis.     

Mesenchymal Deficiency of Notch1 Attenuates Bleomycin-Induced Pulmonary Fibrosis

Notch signaling pathway is involved in the regulation of cell fate, differentiation, proliferation, and apoptosis in development and disease. Previous studies suggest the importance of Notch1 in myofibroblast differentiation in lung alveogenesis and fibrosis. However, direct in vivo evidence of Notch1-mediated myofibroblast differentiation is lacking. In this study, we examined the effects of conditional mesenchymal-specific deletion of Notch1 on pulmonary fibrosis. Crossing of mice bearing the floxed Notch1 gene with α2(I) collagen enhancer-Cre-ER(T)–bearing mice successfully generated progeny with a conditional knockout (CKO) of Notch1 in collagen I–expressing (mesenchymal) cells on treatment with tamoxifen (Notch1 CKO). Because Notch signaling is known to be activated in the bleomycin model of pulmonary fibrosis, control and Notch1 CKO mice were analyzed for their responses to bleomycin treatment. The results showed significant attenuation of pulmonary fibrosis in CKO relative to control mice, as examined by collagen deposition, myofibroblast differentiation, and histopathology. However, there were no significant differences in inflammatory or immune cell influx between bleomycin-treated CKO and control mouse lungs. Analysis of isolated lung fibroblasts confirmed absence of Notch1 expression in cells from CKO mice, which contained fewer myofibroblasts and significantly diminished collagen I expression relative to those from control mice. These findings revealed an essential role for Notch1-mediated myofibroblast differentiation in the pathogenesis of pulmonary fibrosis.Notch signaling is known to play critical roles in development, tissue homeostasis, and disease.^{1, 2, 3, 4, 5, 6, 7, 8, 9, 10} Notch signaling is mediated via four known receptors, Notch 1, 2, 3, and 4, which serve as receptors for five membrane-bound ligands, Jagged 1 and 2 and Delta 1, 3, and 4.^{1, 11, 12, 13} The Notch receptors differ primarily in the number of epidermal growth factor-like repeats and C-terminal sequences.¹³ For instance, Notch 1 contains 36 of epidermal growth factor-like repeats, is composed of approximately 40 amino acids, and is defined largely by six conserved cysteine residues that form three conserved disulfide bonds.^{1, 13, 14, 15} These epidermal growth factor-like repeats can be modified by O-linked glycans at specific sites, which is important for their function.^{1, 14, 15} Modulation of Notch signaling by Fringe proteins,^{16, 17, 18} which are N-acetylglucosamine transferases, illustrates the importance of these carbohydrate residues.^{16, 18} Moreover, mutation of the GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase causes defective fucosylation of Notch1, resulting in impairment of the Notch1 signaling pathway and myofibroblast differentiation.^{19, 20, 21} Because myofibroblasts are important in both lung development and fibrosis, elucidation of the role of Notch signaling in their genesis in vivo will provide insight into the significance of this signaling pathway in either context.The importance of Notch signaling in tissue fibrosis is suggested in multiple studies.^{10, 21, 22, 23, 24} As in other organs or tissues, pulmonary fibrosis is characterized by fibroblast proliferation and de novo emergence of myofibroblasts, which is predominantly responsible for the increased extracellular matrix production and deposition.^{25, 26, 27, 28, 29, 30, 31} Animal models, such as bleomycin-induced pulmonary fibrosis, are characterized by both acute and chronic inflammation with subsequent myofibroblast differentiation that mainly originated from the mesenchymal compartment.^{21, 25, 26, 27, 28} In vitro studies of cultured cells implicate Notch signaling in myofibroblast differentiation,²¹ which is mediated by induction of the Notch1 ligand Jagged1 when lung fibroblasts are treated with found in inflammatory zone 1.²¹ Moreover, GDP-4-keto-6-deoxymannose-3,5-epimerase-4-reductase knockout mice with defective fucosylation of Notch1 exhibit consequent impairment of Notch signaling and attenuated pulmonary fibrosis in studies using the bleomycin model.²¹ The in vivo importance of Notch signaling in myofibroblast differentiation during lung development has also been suggested by demonstration of impaired alveogenesis in mice deficient in lunatic fringe³² or Notch receptors.^{10, 33, 34, 35} These in vivo studies, however, do not pinpoint the cell type in which deficient Notch signaling is causing the observed impairment of myofibroblast differentiation. This is further complicated by the extensive evidence showing that, in addition to myofibroblast differentiation, Notch1 mediates multiple functional responses in diverse cell types, including inflammation and the immune system.^{21, 36, 37, 38} In the case of tissue injury and fibrosis, including the bleomycin model, the associated inflammation and immune response as well as parenchymal injury can affect myofibroblast differentiation via paracrine mechanisms.^{39, 40} Thus, although global impairment of Notch signaling can impair myofibroblast differentiation in vivo, it does not necessarily indicate a specific direct effect on the mesenchymal precursor cell. Furthermore, understanding the importance of Notch signaling in these different cell compartments is critical for future translational studies to develop effective drugs targeting this signaling pathway with minimal off-target or negative adverse effects.In this study, the effects of conditional selective Notch1 deficiency in the mesenchymal compartment on myofibroblast differentiation and bleomycin-induced pulmonary fibrosis were examined using a Cre-Lox strategy. The transgenic Cre mice bore the Cre-ER(T) gene composed of Cre recombinase and a ligand-binding domain of the estrogen receptor⁴¹ driven by a minimal promoter containing a far-upstream enhancer from the α2(I) collagen gene. When activated by tamoxifen, this enhancer enabled selective Cre expression only in type I collagen-expressing (mesenchymal) cells, such as fibroblasts and other mesenchymal cells,⁴² leading to excision of LoxP consensus sequence flanked target gene DNA fragment (floxed gene) of interest.^{41, 43, 44, 45, 46} To evaluate the importance of Notch1 in the mesenchymal compartment and discriminate its effects from those in the inflammatory and immune system and other compartments, the transgenic Cre-ER(T) mice [Col1α2-Cre-ER(T)^+/0] were crossed with mice harboring the floxed (containing loxP sites) Notch1 gene (Notch1^fl/fl). The resulting progeny mice [Notch1 conditional knockout (CKO)] that were homozygous for the floxed Notch1 allele and hemizygous for the Col1α2-Cre-ER(T) allele with genotype [Notch1^fl/fl,Col1α2-Cre-ER(T)^+/0] were Notch1 deficient in the mesenchymal compartment when injected with tamoxifen. Control Notch1 wild-type (WT) mice exhibited the expected pulmonary fibrosis along with induction of Jagged1 and Notch1 on treatment with bleomycin, consistent with previous observation of Notch signaling activation in this model.²¹ Isolated and cultured Notch1 CKO mouse lung fibroblasts were deficient in Notch1 and exhibited diminished myofibroblast differentiation compared with cells from the corresponding WT control mice. Most important, compared with WT control mice, the CKO mice exhibited diminished bleomycin-induced pulmonary fibrosis that was accompanied by significant reduction in α-smooth muscle actin (α-SMA) and type I collagen gene expression, consistent with defective myofibroblast differentiation. In contrast, enumeration of lung inflammatory and immune cells failed to show a significant difference in bleomycin-induced recruitment of these cells between control and CKO mice. Thus, selective Notch1 deficiency in mesenchymal cells caused impairment of fibrosis that is at least, in part, because of deficient myofibroblast differentiation, and without affecting the inflammatory and immune response in this animal model.     

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.Although fat necrosis has been associated with severe cases of pancreatitis for more than a century,^{1, 2} and alcohol consumption is a well-known risk factor for acute pancreatitis (AP),³ only recently have we started understanding the mechanistic basis of these observations.^{4, 5, 6, 7} High amounts of unsaturated fatty acids (UFAs) have been noted in the pancreatic necrosis and sera of severe AP (SAP) patients by multiple groups.^{8, 9, 10, 11, 12} These high UFAs seem pathogenically relevant because several studies show UFAs can cause pancreatic acinar injury or can worsen AP.^{11, 12, 13, 14} Ethanol may play a role in AP by distinct mechanisms,³ including a worse inflammatory response to cholecystokinin,⁴ increased zymogen activation,¹⁵ basolateral enzyme release,¹⁶ sensitization to stress,⁷ FA ethyl esters (FAEEs),¹⁷ cytosolic calcium,¹⁸ and cell death.¹⁹Because the nonoxidative ethanol metabolite of fatty acids (FAs), FAEEs, were first noted to be elevated in the pancreata of dying alcoholics, they have been thought to play a role in AP.^{17, 19, 20, 21, 22} Conclusive proof of the role of FAEEs in AP in comparison with their parent UFAs is lacking. Uncontrolled release of lipases into fat, whether in the pancreas or in the peritoneal cavity, may result in fat necrosis, UFA generation, which has been associated with SAP.^{11, 12} Pancreatic homogenates were also noted to have an ability to synthesize FAEEs from FAs and ethanol,^{20, 23} and the putative enzyme for this was thought to be a lipase.^{24, 25} It has been shown that the FAEE synthase activity of the putative enzyme exceeds its lipolytic capacity by several fold.²⁵Triglyceride (TG) forms >80% of the adipocyte mass,^{26, 27, 28} oleic acid (OA) being the most enriched FA.^{9, 29} We recently showed that lipolysis of intrapancreatic TG worsens pancreatitis.^{11, 12} Therefore, after noting the ability of the pancreas to cause lipolysis of TG into FAs and also to have high FAEE synthase activity and FAEE concentrations, we decided to compare the relative ability of FAEEs and their parent FAs to initiate deleterious signaling in pancreatitis and to investigate their impact on the severity of AP.     

Type I Interferon Contributes to Noncanonical Inflammasome Activation,Mediates Immunopathology,and Impairs Protective Immunity during Fatal Infection with Lipopolysaccharide-Negative Ehrlichiae

Ehrlichia species are intracellular bacteria that cause fatal ehrlichiosis, mimicking toxic shock syndrome in humans and mice. Virulent ehrlichiae induce inflammasome activation leading to caspase-1 cleavage and IL-18 secretion, which contribute to development of fatal ehrlichiosis. We show that fatal infection triggers expression of inflammasome components, activates caspase-1 and caspase-11, and induces host-cell death and secretion of IL-1β, IL-1α, and type I interferon (IFN-I). Wild-type and Casp1^−/− mice were highly susceptible to fatal ehrlichiosis, had overwhelming infection, and developed extensive tissue injury. Nlrp3^−/− mice effectively cleared ehrlichiae, but displayed acute mortality and developed liver injury similar to wild-type mice. By contrast, Ifnar1^−/− mice were highly resistant to fatal disease and had lower bacterial burden, attenuated pathology, and prolonged survival. Ifnar1^−/− mice also had improved protective immune responses mediated by IFN-γ and CD4⁺ Th1 and natural killer T cells, with lower IL-10 secretion by T cells. Importantly, heightened resistance of Ifnar1^−/− mice correlated with improved autophagosome processing, and attenuated noncanonical inflammasome activation indicated by decreased activation of caspase-11 and decreased IL-1β, compared with other groups. Our findings demonstrate that IFN-I signaling promotes host susceptibility to fatal ehrlichiosis, because it mediates ehrlichia-induced immunopathology and supports bacterial replication, perhaps via activation of noncanonical inflammasomes, reduced autophagy, and suppression of protective CD4⁺ T cells and natural killer T-cell responses against ehrlichiae.Ehrlichia chaffeensis is the causative agent of human monocytotropic ehrlichiosis, a highly prevalent life-threatening tickborne disease in North America.^{1, 2, 3} Central to the pathogenesis of human monocytotropic ehrlichiosis is the ability of ehrlichiae to survive and replicate inside the phagosomal compartment of host macrophages and to secrete proteins via type I and type IV secretion systems into the host-cell cytosol.⁴ Using murine models of ehrlichiosis, we and others have demonstrated that fatal ehrlichial infection is associated with severe tissue damage caused by TNF-α–producing cytotoxic CD8⁺ T cells (ie, immunopathology) and the suppression of protective CD4⁺ Th1 immune responses.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14} However, neither how the Ehrlichia bacteria trigger innate immune responses nor how these responses influence the acquired immunity against ehrlichiae is entirely known.Extracellular and intracellular pattern recognition receptors recognize microbial infections.^{15, 16, 17, 18} Recently, members of the cytosolic nucleotide-binding domain and leucine-rich repeat family (NLRs; alias NOD-like receptors), such as NLRP3, have emerged as critical pattern recognition receptors in the host defense against intracellular pathogens. NLRs recognize intracellular bacteria and trigger innate, protective immune responses.^{19, 20, 21, 22, 23} NLRs respond to both microbial products and endogenous host danger signals to form multimeric protein platforms known as inflammasomes. The NLRP3 inflammasome consists of multimers of NLRP3 that bind to the adaptor molecules and apoptosis-associated speck-like protein (ASC) to recruit pro–caspase-1 and facilitate cleavage and activation of caspase-1.^{15, 16, 24} The canonical inflammasome pathway involves the cleavage of immature forms of IL-1β and IL-18 (pro–IL-1β and pro–IL-18) into biologically active mature IL-1β and IL-18 by active caspase-1.^{25, 26, 27, 28} The noncanonical inflammasome pathway marked by the activation of caspase-11 has been described recently. Active caspase-11 promotes the caspase-1–dependent secretion of IL-1β/IL-18 and mediates inflammatory lytic host-cell death via pyroptosis, a process associated with the secretion of IL-1α and HMGB1.^{17, 29, 30, 31} Several key regulatory checkpoints ensure the proper regulation of inflammasome activation.^{16, 32} For example, blocking autophagy by the genetic deletion of the autophagy regulatory protein ATG16L1 increases the sensitivity of macrophages to the inflammasome activation induced by TLRs.³³ Furthermore, TIR domain-containing adaptor molecule 1 (TICAM-1; alias TRIF) has been linked to inflammasome activation via the secretion of type I interferons α and β (IFN-α and IFN-β) and the activation of caspase-11 during infections with Gram-negative bacteria.^{2, 34, 35, 36, 37, 38, 39}We have recently demonstrated that fatal ehrlichial infection induces excess IL-1β and IL-18 production, compared with mild infection,^{8, 12, 13, 14} and that lack of IL-18 signaling enhances resistance of mice to fatal ehrlichiosis.¹² These findings suggest that inflammasomes play a detrimental role in the host defense against ehrlichial infection. Elevated production of IL-1β and IL-18 in fatal ehrlichiosis was associated with an increase in hepatic expression of IFN-α.¹⁴ IFN-I plays a critical role in the host defense against viral and specific bacterial infections.^{28, 36, 37, 40, 41, 42, 43} However, the mechanism by which type I IFN contributes to fatal ehrlichial infection remains unknown. Our present results reveal, for the first time, that IFNAR1 promotes detrimental inflammasome activation, mediates immunopathology, and impairs protective immunity against ehrlichiae via mechanisms that involve caspase-11 activation, blocking of autophagy, and production of IL-10. Our novel finding that lipopolysaccharide (LPS)-negative ehrlichiae trigger IFNAR1-dependent caspase-11 activation challenges the current paradigm that implicates LPS as the major microbial ligand triggering the noncanonical inflammasome pathway during Gram-negative bacterial infection.     

Reduced Innervation and Delayed Re-Innervation After Epithelial Wounding in Type 2 Diabetic Goto-Kakizaki Rats

Patients with diabetes are at an increased risk for developing corneal complications including delayed wound healing and potential vision loss. To understand the cause of diabetic keratopathy, we investigated innervation and its correlation with delayed corneal epithelial wound healing in type 2 diabetic Goto-Kakizaki (GK) rats. GK rats are smaller than the age-matched control Wistar rats from which the GK rats were derived. The blood sugar levels of GK rats are significantly higher than those of Wistar rats. GK rats had increased rose bengal staining and cornea fragility. Fewer nerve fibers were detected compared with Wistar rats. Although nerve fiber densities detected by whole-mount immunohistochemistry were similar near the limbal region, in the central cornea the subbasal nerve plexuses were thinner, less abundant, and showed less branching in GK rats. Corneal epithelial wound closure was delayed and re-innervation was slow and incomplete in GK rats. These abnormalities were more apparent in older GK rats (12 months). Our data suggest that diabetic neuropathy occurs in the cornea of type 2 diabetic GK rats, and defects in the sensory nerve and/or tear film may contribute to diabetic keratopathy and delayed epithelial wound healing in diabetic corneas.With the rapid increase in the prevalence of diabetes mellitus (DM), mostly in type 2 DM, ocular complications have become a leading cause of blindness in the world.¹ In addition to abnormalities of the retina (diabetic retinopathy) and the lens (cataract), various types of ocular mucosal surface disorders are also relatively common in DM patients.² They include impaired corneal sensation,^3–6 reduced tear secretion,^7,8 conjunctival squamous metaplasia and goblet cell loss,⁹ and corneal keratopathy.^2,3 Diabetic keratopathy occurs in more than 70% of diabetic patients^2–4 and increases the susceptibility of the cornea to trauma with epithelial erosions and ulcerations.^5,6,10 Although the cornea is an avascular tissue, it is the most densely innervated part of the human body, containing Aδ and unmyelinated C fibers, and derives its innervation from the ophthalmic division of the trigeminal nerve.¹¹ The sensory nerve fibers in diabetic patients with peripheral neuropathy probably undergo the earliest damage in diabetes.^12,13 Therefore, the aforementioned abnormalities also can be attributed to the decrease or loss of the nerve ends and fibers, and diabetic keratopathy also can be thought of as a form of neuropathy.^11–13 As a matter of fact, because corneal nerve structure and function can be assessed readily and accurately using in vivo corneal confocal microscopy and noncontact corneal esthesiometry, respectively, assessing corneal neuropathy has been proposed as a noninvasive and reliable way to diagnose peripheral diabetic neuropathy (keratopathy), a debilitating condition that affects approximately 50% of diabetic patients.¹⁴The cornea is a transparent tissue consisting of three cellular layers: the epithelium, stroma, and a simple epithelial layer, termed endothelium. Many major hyperglycemia-caused pathologic changes in the cornea occur around the stratified epithelia, including alterations in the epithelial basement membrane such as thickening,^6,15 a decreased number of hemidesmosomes,¹⁶ and the deposition of advanced glycation end products.^6,17 Hyperglycemia also directly affects epithelial cells and significantly alters its structure and function, resulting in basal cell degeneration,^10,18,19 decreased^20,21 or increased²² cell proliferation, superficial punctate keratitis,²³ breakdown of barrier function,^24,25 and fragility,²⁶ depending on the duration of DM and on the serum concentration of glycated hemoglobin HbA_1c. Hence, diabetic keratopathy also was termed “diabetic corneal epitheliopathy.”^27,28 Clinically, the cornea appears normal in patients with diabetic keratopathy in the absence of corneal injury. However, trauma and ocular surgeries, such as vitrectomy for vitreous hemorrhage, may require the removal of epithelial cells or damage the fragile structure, causing cell injury and/or removal of corneal epithelium. In diabetic patients, there is a considerable delay in corneal re-epithelialization after injury. The impairment of corneal epithelial wound healing could result in several types of epithelial disorders, such as persistent epithelial defects and recurrent erosion in patients after surgery.^2,29–32 Furthermore, delayed healing of the epithelial defect may be associated with sight-threatening complications, such as stromal opacification, surface irregularity, and microbial keratitis.²⁹ Hence, a better understanding of the mechanisms underlying delayed epithelial wound healing in diabetic corneas should lead to better management of the disease.We previously used a streptozocin (STZ)-induced rat model of type 1 diabetes and showed that delayed wound healing in diabetic rats is associated with the impairment of epidermal growth factor receptor–mediated cell signaling in response to mechanical injury.^21,25,28 These STZ rats had stronger rose bengal staining, decreased tear secretion, slightly attenuated sensitivity, and reduced nerve fibers. To better understand diabetic keratopathy in type 2 DM (T2D), which develops as a result of a failure to increase β-cell function and mass adequately to meet the demands of prevailing insulin resistance,³³ we maintained a colony of Goto-Kakizaki (GK) rats, one of the best characterized animal models of spontaneous T2D that were derived from Wistar rats.^34,35 We noticed that, unlike Sprague-Dawley (SD) rats, Wistar rats were less sensitive to aesthesiometer with thread and had reduced corneal sensitivity. In this study, we characterized the ocular alterations of GK rats. Our study revealed that, in addition to changes in nerve fibers, the subbasal nerve ends also were decreased. This decrease in corneal innervation may contribute to tear deficiency and delayed wound healing in GK rats.     

A Tissue-Specific Role for Nlrp3 in Tubular Epithelial Repair after Renal Ischemia/Reperfusion

Ischemia/reperfusion injury is a major cause of acute kidney injury. Improving renal repair would represent a therapeutic strategy to prevent renal dysfunction. The innate immune receptor Nlrp3 is involved in tissue injury, inflammation, and fibrosis; however, its role in repair after ischemia/reperfusion is unknown. We address the role of Nlrp3 in the repair phase of renal ischemia/reperfusion and investigate the relative contribution of leukocyte- versus renal-associated Nlrp3 by studying bone marrow chimeric mice. We found that Nlrp3 expression was most profound during the repair phase. Although Nlrp3 expression was primarily expressed by leukocytes, both leukocyte- and renal-associated Nlrp3 was detrimental to renal function after ischemia/reperfusion. The Nlrp3-dependent cytokine IL-1β remained unchanged in kidneys of all mice. Leukocyte-associated Nlrp3 negatively affected tubular apoptosis in mice that lacked Nlrp3 expression on leukocytes, which correlated with reduced macrophage influx. Nlrp3-deficient (Nlrp3KO) mice with wild-type bone marrow showed an improved repair response, as seen by a profound increase in proliferating tubular epithelium, which coincided with increased hepatocyte growth factor expression. In addition, Nlrp3KO tubular epithelial cells had an increased repair response in vitro, as seen by an increased ability of an epithelial monolayer to restore its structural integrity. In conclusion, Nlrp3 shows a tissue-specific role in which leukocyte-associated Nlrp3 is associated with tubular apoptosis, whereas renal-associated Nlrp3 impaired wound healing.Ischemia/reperfusion (IR) injury is a major cause of acute kidney injury¹ and increases the risk of developing chronic kidney disease (CKD).² After injury, wounded tissue organizes an efficient response that aims to combat infections, clear cell debris, re-establish cell number, and reorganize tissue architecture. First, necrotic tissue releases danger-associated molecular patterns, such as high-mobility group box-1³ or mitochondrial DNA,⁴ which leads to chemokine secretion⁵ and a subsequent influx of leukocytes. Second, neutrophils and macrophages clear cellular debris but also increase renal damage because depletion of neutrophils⁶ or macrophages within 48 hours of IR will reduce renal damage.⁷ At approximately 72 hours of reperfusion, the inflammatory phase transforms into the repair phase and is characterized by surviving tubular epithelial cells (TECs) that dedifferentiate, migrate, and proliferate to restore renal function.⁸Previously, we have shown that Toll-like receptor (TLR) 2 and TLR4 play a detrimental role after acute renal IR injury.^{9, 10, 11} In addition, TLR2 appeared also pivotal in mediating tubular repair in vitro after cisplatin-induced injury,¹² indicating a dual role for TLR2. The cytosolic innate immune receptor Nlrp3 is able to sense cellular damage¹³ and mediates renal inflammation and pathological characteristics after IR^{14, 15, 16} or nephrocalcinosis.¹⁷ Next to the detrimental role of Nlrp3 in different renal disease models and consistent with the dual role of TLR2, Nlrp3 was shown to protect against loss of colonic epithelial integrity.¹⁸ We, therefore, speculate that Nlrp3, which contributes to sterile renal inflammation during acute renal IR injury, might also drive subsequent tubular repair.To test this hypothesis, we investigated the role of leukocyte- versus renal-associated Nlrp3 with respect to tissue repair after renal IR. We observed that both renal- and leukocyte-associated Nlrp3s are detrimental to renal function after renal IR injury; however, this is through different mechanisms. Leukocyte-associated Nlrp3 is related to increased tubular epithelial apoptosis, whereas renal-associated Nlrp3 impairs the tubular epithelial repair response. Our data suggest Nlrp3 as a negative regulator of resident tubular cell proliferation in addition to its detrimental role in renal fibrosis and inflammation.^{14, 19}     

Synaptic Amyloid-β Oligomers Precede p-Tau and Differentiate High Pathology Control Cases

Amyloid-β (Aβ) and hyperphosphorylated tau (p-tau) aggregates form the two discrete pathologies of Alzheimer disease (AD), and oligomeric assemblies of each protein are localized to synapses. To determine the sequence by which pathology appears in synapses, Aβ and p-tau were quantified across AD disease stages in parietal cortex. Nondemented cases with high levels of AD-related pathology were included to determine factors that confer protection from clinical symptoms. Flow cytometric analysis of synaptosome preparations was used to quantify Aβ and p-tau in large populations of individual synaptic terminals. Soluble Aβ oligomers were assayed by a single antibody sandwich enzyme-linked immunosorbent assay. Total in situ Aβ was elevated in patients with early- and late-stage AD dementia, but not in high pathology nondemented controls compared with age-matched normal controls. However, soluble Aβ oligomers were highest in early AD synapses, and this assay distinguished early AD cases from high pathology controls. Overall, synapse-associated p-tau did not increase until late-stage disease in human and transgenic rat cortex, and p-tau was elevated in individual Aβ-positive synaptosomes in early AD. These results suggest that soluble oligomers in surviving neocortical synaptic terminals are associated with dementia onset and suggest an amyloid cascade hypothesis in which oligomeric Aβ drives phosphorylated tau accumulation and synaptic spread. These results indicate that antiamyloid therapies will be less effective once p-tau pathology is developed.A large body of evidence indicates that soluble oligomers of amyloid-β (Aβ) are the primary toxic peptides that initiate downstream tau pathology in the amyloid cascade hypothesis of Alzheimer disease (AD).^{1, 2} However, the time course and severity of AD dementia have been generally found to correlate with neurofibrillary tangle development rather than plaque appearance,^{3, 4, 5, 6, 7, 8} although a few studies have linked plaques with early cognitive decline.^{9, 10, 11, 12} Soluble oligomeric Aβ has been highlighted as the primary toxin for loss of dendritic spines and synaptic function¹³ and has also been directly linked to downstream tau pathology. For example, suppression of a tau kinase pathway can prevent Aβ42 oligomer-induced dendritic spine loss,¹⁴ and injection of Aβ42 fibrils into mutant tau mice induces neurofibrillary tangles in cell bodies retrograde to the injections.¹⁵ In vivo, effects of Aβ oligomers versus fibrils are harder to separate; however, lowering soluble Aβ oligomers by halving β–site amyloid precursor protein (APP) cleaving enzyme reduces accumulation and phosphorylation of wild-type tau in a mouse model.¹⁶ Evidence for Aβ and tau association is particularly strong in the dendritic compartment, where tau was shown to mediate Aβ toxicity via linkage of fyn to downstream N-methyl-d-aspartate receptor toxicity.¹⁷The earliest cognitive losses in AD have long been thought to correlate with synapse loss.^{8, 18, 19, 20, 21} In humans, electron microscopic studies have documented synapse-associated Aβ and tau,^{22, 23} and much work documents activity-dependent release of synaptic Aβ into interstitial fluid, which drives local Aβ deposition in human subjects and in rodents.^{4, 24, 25} Of importance, most synapse-associated Aβ in cortical synapses of AD patients consists of soluble oligomeric species,²⁶ and synaptic tau pathology in AD also includes accumulations of SDS-stable tau oligomers.^{27, 28, 29, 30, 31} With the use of synaptosomes (resealed nerve terminals) from the cortex of postmortem human subjects and a transgenic rat model of AD, the present experiments were aimed at determining the sequence of appearance of Aβ and hyperphosphorylated tau (p-tau) pathology in synaptic terminals. In addition to early- and late-stage disease, the AD samples included nondemented high pathology controls (HPCs) with substantial AD-related pathology. Synaptic accumulation of Aβ occurred in the earliest plaque stages, before the appearance of synaptic p-tau, which did not appear until late-stage disease. Soluble Aβ oligomers in synaptic terminals were elevated in early AD cases compared with HPCs, indicating an association with the onset of a dementia diagnosis.     

CUL4high Lung Adenocarcinomas Are Dependent on the CUL4-p21 Ubiquitin Signaling for Proliferation and Survival

Cullin (CUL) 4A and 4B ubiquitin ligases are often highly accumulated in human malignant neoplasms and are believed to possess oncogenic properties. However, the underlying mechanisms by which CUL4A and CUL4B promote pulmonary tumorigenesis remain largely elusive. This study reports that CUL4A and CUL4B are highly expressed in patients with non–small cell lung cancer (NSCLC), and their high expression is associated with disease progression, chemotherapy resistance, and poor survival in adenocarcinomas. Depletion of CUL4A (CUL4A^k/d) or CUL4B (CUL4B^k/d) leads to cell cycle arrest at G₁ and loss of proliferation and viability of NSCLC cells in culture and in a lung cancer xenograft model, suggesting that CUL4A and 4B are oncoproteins required for tumor maintenance of certain NSCLCs. Mechanistically, increased accumulation of the cell cycle–dependent kinase inhibitor p21/Cip1/WAF1 was observed in lung cancer cells on CUL4 silencing. Knockdown of p21 rescued the G₁ arrest of CUL4A^k/d or CUL4B^k/d NSCLC cells, and allowed proliferation to resume. These findings reveal that p21 is the primary downstream effector of lung adenocarcinoma dependence on CUL4, highlight the notion that not all substrates respond equally to abrogation of the CUL4 ubiquitin ligase in NSCLCs, and imply that CUL4A^high/CUL4B^high may serve as a prognostic marker and therapeutic target for patients with NSCLC.

Lung cancer is the most common cause of cancer mortality worldwide,¹ accounting for 19.4% of all cancer-related deaths and representing a significant clinical burden.² Among the subtypes of lung cancer, non–small cell lung cancer (NSCLC) accounts for 80% to 85% of cases.3, 4, 5 Although multimodality treatments, including targeted therapies and immunotherapies, have been applied to NSCLCs, with high rates of local and distant failure, the overall cure and survival rates for NSCLC remain low.^6,7 Thus, understanding the molecular mechanisms underlying NSCLC development and progression is of fundamental importance for the development of new therapeutic strategies for patients with NSCLC.Cullin (CUL) 4, a molecular scaffold of the CUL4-RING ubiquitin ligase (CRL4), plays an important role in regulating key cellular processes through modulating the ubiquitylation and degradation of various protein substrates.⁸ Two CUL4 proteins, CUL4A and CUL4B, share an 82% sequence homology, with similar but distinct functions.⁹ CUL4 has been extensively studied in the process of nucleotide excision repair (NER) after UV irradiation.10, 11, 12, 13 Loss of CUL4A, but not CUL4B, elevates global genomic NER activity and confers increased protection against UV-induced skin carcinogenesis.¹¹ In addition to DNA repair, CUL4 also plays a significant role in a wide spectrum of physiologic processes, such as the cell cycle, cell signaling, and histone methylation, which have direct relevance to the development of human cancers.14, 15, 16 Accumulating studies have found that CUL4A is amplified or expressed at abnormally high levels in multiple cancers, including breast cancer, squamous cell carcinoma, hepatocellular carcinomas, and lung cancer.^9,17, 18, 19 More importantly, CUL4A and 4B overexpression is implicated in tumor progression, metastasis, and a poorer survival rate for patients with cancer.^9,20,21 CUL4A, but not CUL4B, is inversely correlated with the NER protein xeroderma pigmentosum, complementation group C and the G₁/S DNA damage checkpoint protein p21 in patients with lung squamous cell carcinoma, highlighting a reduced DNA damage response⁹ as well as promoting cell growth and tumorigenesis.^22,23 Increased expression of CUL4A caused hyperplasia as well as lung adenocarcinomas in mice.²⁴ However, the mechanistic basis and clinical significance of CUL4A dysregulation in NSCLC remain unclear.The CUL4A paralog CUL4B shares extensive sequence homology and redundant functions with CUL4A.⁹ To date, research on CUL4B has been focused mainly on its genetic association with human X-linked mental retardation.25, 26, 27, 28 Recently, CUL4B was found to be overexpressed in colon cancer and correlated with tumor stage, histologic differentiation, vascular invasion, and distant metastasis.²⁹ Patients with lung and colon cancer with high levels of CUL4B had lower overall survival (OS) and disease-free survival (DFS) rates than those with low CUL4B expression.^9,29 CUL4B is also overexpressed in cervical, esophageal, and breast cancers and associated with tumor invasion and lymph node metastasis.^16,30,31 Furthermore, CUL4B overexpression promotes the development of spontaneous liver tumors at a high rate and enhances diethylnitrosamine-induced hepatocarcinogenesis in transgenic mice.³²The molecular mechanisms underlying the capacity of CUL4 to promote pulmonary tumorigenesis remain largely elusive. CUL4A promotes NSCLC cell growth.²² CUL4 targets a panel of cell cycle regulators for ubiquitination and degradation, including Cdc6, Cdt1, p21, cyclin E, minichromosome maintenance 10 replication initiation factor, and forkhead box M1.³³ However, which of the cell cycle substrates of CUL4 play a key role in tumor dependence on dysregulated CUL4A or CUL4B remains to be defined. This study found that attenuation of CUL4, especially CUL4B, inhibited NSCLC cell proliferation and tumorigenesis through increased accumulation of p21 and cell cycle arrest in G₁.     

p53 Mediates TNF-Induced Epithelial Cell Apoptosis in IBD

Chronic ulcerative colitis (CUC) is characterized by increased intestinal epithelial cell (IEC) apoptosis associated with elevated tumor necrosis factor (TNF), inducible nitric oxide synthase (iNOS), and p53. We previously showed that p53 is increased in crypt IECs in human colitis and is needed for IEC apoptosis in chronic dextran sulfate sodium-colitis. Herein, we examined the roles of TNF and iNOS in regulating p53-induced IEC apoptosis in CUC. The IEC TUNEL staining, caspases 3, 8, and 9, and p53 protein levels, induced by anti-CD3 monoclonal antibody (mAb) activation of T cells, were markedly reduced in TNF receptor 1 and 2 gene knockout mice. Induction of IEC apoptosis correlated with increased p53, which was attenuated in iNOS^−/− mice. IEC p53 levels and apoptosis were reduced in IL-10^−/− colitic mice treated with neutralizing TNF mAb and the iNOS inhibitor, aminoguanidine, further suggesting that TNF and iNOS are upstream of p53 during colitis-induced IEC apoptosis. IEC apoptosis and p53 levels were assessed in control versus untreated or anti-TNF–treated CUC patients with equivalent levels of inflammation. Data indicated that IEC apoptosis and p53 levels were clearly higher in untreated CUC but markedly reduced in patients treated with anti-TNF mAb. Therefore, TNF-induced iNOS activates a p53-dependent pathway of IEC apoptosis in CUC. The inhibition of IEC apoptosis may be an important mechanism for mucosal healing in anti-TNF–treated CUC patients.Human inflammatory bowel diseases (IBDs) are characterized by excessive crypt epithelial apoptosis, surface ulceration, distorted crypt architecture, diarrhea, and bleeding. Barrier disruption is linked to epithelial apoptosis caused by aberrant activation of innate and adaptive immune responses.^1–3 A hallmark of severe IBD is the overproduction of tumor necrosis factor (TNF) in mucosal tissue.^2,4 The importance of TNF in disease pathogenesis is underlined by the pronounced clinical improvement induced when anti-TNF antibodies reduce diarrhea, weight loss, and bleeding.^4,5 At the mucosal level, anti-TNF antibody treatment enhances mucosal healing with rapid re-epithelialization of ulcerated surfaces. Studies indicate some (eg, infliximab and adalimumab), but not all (eg, certolizumab), anti-TNF agents induce apoptosis of lamina propria cells, despite all three being able to enhance mucosal healing.^6,7 However, it remains unclear whether the effect of anti-TNF on mucosal healing is related to reduced epithelial apoptosis and, if so, through what mechanism.Overproduction of TNF in IBD has potent effects on mucosal adaptive and innate immune responses.^8,9 TNF participates in macrophage activation by enhancing antimicrobial functions.¹⁰ In response to TNF, macrophages increase production of reactive nitrosative species, such as nitric oxide (NO⁻) and its metabolite, peroxynitrite (ONOO⁻).¹¹ Inducible NO synthase (iNOS) blockade inhibits disease severity and epithelial apoptosis in animal models of IBD.^12,13 Data from human IBD studies suggest that NO⁻ and ONOO⁻ stabilize p53 and activate response pathways.^14,15 During tumorigenesis, NO⁻-induced mutations of p53 inactivate tumor suppressor function, with loss of protective effects.¹⁶ Thus, TNF-mediated activation of iNOS may be an important pathway for regulating epithelial cell apoptosis during colitis and colitis-induced dysplasia.Understanding TNF receptor signaling is complex and difficult to apply to in vivo systems. TNF receptor 1 (TNFR1) associates with the TNF receptor–associated death domain, which activates the extrinsic, caspase 8–linked pathway of apoptosis.^17,18 However, in some systems examined, TNF receptor–associated death domain is dispensable for TNF-induced apoptosis, and cross activation of TNFRl and TNFR2 converges unto common downstream signaling events, resulting in apoptosis mediated by intrinsic (mitochondrial) pathways.^17,19 The proliferative zone for intestinal epithelial cells (IECs) resides in lower crypt regions. Cellular proliferation requires enhanced mitochondrial function. Given that epithelial apoptosis in IBD occurs in proliferative crypt epithelial cells, we suspected that pathways involving induction of mitochondrial pathways were used. In addition, a comprehensive understanding of the role of TNF receptor signaling within the mucosal microenvironment requires that receptor deficiency be restricted to distinct populations participating in mucosal immune responses.Increased epithelial crypt cell apoptosis commonly occurs in ulcerative colitis (UC) and Crohn''s disease.^20,21 Numerous in vitro and in vivo model systems have studied this phenomenon, suggesting that TNF-mediated pathways play key roles in inducing programmed cell death in epithelial crypts. To model these pathways, a well-characterized model of T-cell activation was used that induces transient stem/progenitor cell activation, crypt IEC proliferation, and TNF-mediated diarrhea reminiscent of human IBD.^22,23 The reproducible kinetics of the model permitted identification of the events in immune-mediated apoptosis and allowed application to relevant gene knockout models. We recently reported that p53 is the major mediator of colonic crypt IEC apoptosis in colitis.²⁴ This article examines the upstream events leading up to p53 activation and IEC apoptosis. Results suggest a mechanism by which TNF signals, through both TNFR1 and TNFR2, stimulate iNOS-mediated p53-dependent apoptosis of crypt IECs. Studies in the IL-10^−/− murine model of colitis confirmed that TNF-induced iNOS led to activation of p53 and induced IEC apoptosis. Finally, we confirm that TNF-induced p53-mediated apoptosis also occurs in vivo during human UC. Overall, the findings suggest that T-cell activation causes TNF and iNOS-mediated stabilization of p53, followed by p53-mediated crypt cell apoptosis in IBD. These data have direct relevance to mechanisms of barrier disruption, ulceration, and initiation of dysplasia seen in p53 mutant crypts.     

Knockdown of Osteopontin Reduces the Inflammatory Response and Subsequent Size of Postsurgical Adhesions in a Murine Model

Adhesions between organs after abdominal surgery remain a significant unresolved clinical problem, causing considerable postoperative morbidity. Osteopontin (OPN) is a cytokine up-regulated after cell injury and tissue repair. Our previous studies have shown that blocking OPN expression at sites of cutaneous wounding resulted in reduced granulation tissue and scarring. We hypothesize that it may be possible to similarly reduce inflammation-associated fibrosis that causes small-bowel adhesions after abdominal surgery. By using a mouse model, we deliver OPN antisense oligodeoxynucleotides via Pluronic gel to the surface of injured, juxtaposed small bowel and show a significant reduction of inflammatory cell influx to the developing adhesion and a dramatic reduction in the resulting adhesion size. A significant reduction in α-smooth muscle actin expression and collagen deposition within the mature adhesion is also demonstrated. We see no impact on mortality, and the healing of serosal injury to intact bowel appeared normal given the reduced inflammatory response. Our studies suggest that dampening OPN levels might be a potentially important target for anti-adhesion therapeutics.The peritoneum is an extensive and complex organ consisting of a layer of mesothelial cells lining the peritoneal cavity and all organs within it.¹ One of the main functions of the peritoneum is to allow friction-free movement between abdominal viscera and the peritoneal wall.² Any surgery that breaches the peritoneal lining causes injury to the peritoneum, which responds by raising inflammatory signals that attract innate immune cells in parallel with a wound repair response and subsequent fibrosis.^3–5 This almost invariably results in permanent peritoneal adhesion formation.⁶ The result can be tethering of adjacent small-bowel loops that may lead to abdominal pain⁷ and/or bowel obstruction,⁸ which is a significant cause of postoperative morbidity in clinical practice. Readmission rates secondary to adhesional complications are as high as 5% to 10% after abdominal surgery.^9,10 Adhesion prevention options in clinical practice are limited to either barrier methods¹¹ or flotation fluids,¹² which use the concept of keeping damaged peritoneal surfaces separated during their healing process; however, these options are of limited effectiveness.^13,14 Pathophysiological manipulation of the cascade events leading to fibrosis has been investigated,^15–18 but none has led to a clinically usable product. Herein, we investigate whether therapeutic strategies used to block scar formation after skin healing might also be effective during peritoneal repair. Microarray studies of wound tissues from wild-type mice versus PU.1 mice (lacking neutrophils, macrophages, and mast cells) reveal an inflammation-dependent gene, osteopontin (OPN), that is expressed by wound granulation tissue fibroblasts, coincident with a skin wound inflammatory response.^19,20 PU.1 mice heal skin wounds without the standard inflammatory cascade, which results in less fibrosis and scarring at the healed wound site.¹⁹ OPN acts both as a secreted chemokine-like protein and as part of an intracellular signaling complex.²¹ It plays key roles in several processes associated with tissue repair, including cell adhesion, migration, and survival.^21,22 Short-term local knockdown of OPN in cutaneous wounds leads to decreased granulation tissue and reduced scar formation.²³ In this study, we investigate whether these effects are transferable to peritoneal repair and also might block i.p. fibrosis.     

Retinal Dystrophy and Optic Nerve Pathology in?the Mouse Model of Mucolipidosis IV

Mucolipidosis IV is a debilitating developmental lysosomal storage disorder characterized by severe neuromotor retardation and progressive loss of vision, leading to blindness by the second decade of life. Mucolipidosis IV is caused by loss-of-function mutations in the MCOLN1 gene, which encodes the transient receptor potential channel protein mucolipin-1. Ophthalmic pathology in patients includes corneal haze and progressive retinal and optic nerve atrophy. Herein, we report ocular pathology in Mcoln1^−/− mouse, a good phenotypic model of the disease. Early, but non-progressive, thinning of the photoreceptor layer, reduced levels of rhodopsin, disrupted rod outer segments, and widespread accumulation of the typical storage inclusion bodies were the major histological findings in the Mcoln1^−/− retina. Electroretinograms showed significantly decreased functional response (scotopic a- and b-wave amplitudes) in the Mcoln1^−/− mice. At the ultrastructural level, we observed formation of axonal spheroids and decreased density of axons in the optic nerve of the aged (6-month-old) Mcoln1^−/− mice, which indicates progressive axonal degeneration. Our data suggest that mucolipin-1 plays a role in postnatal development of photoreceptors and provides a set of outcome measures that can be used for ocular therapy development for mucolipidosis IV.Mucolipidosis type IV (MLIV) is an autosomal recessive disease characterized by severe psychomotor retardation and visual loss. MLIV is classified as a lysosomal storage disease because of abnormal accumulation of storage material in lysosomes of all cells and tissues of the body.¹ Corneal clouding because of accumulation of lysosomal storage is an early pathological hallmark of the disease that, when present with developmental delay in infanthood, is highly suggestive of MLIV.Ophthalmic manifestations in patients generally have a progressive course and, in addition to corneal clouding, include optic nerve atrophy and outer retinal degeneration.^{2, 3, 4} In most of the patients, MLIV leads to blindness in the second decade of life.⁵Mutations in MCOLN1, which encodes the transient receptor potential cation channel TRPML1 (alias mucolipin-1) cause the disease.^{6, 7, 8, 9} More than 20 mutations in MCOLN1 have been identified to date.⁵ More than 75% of known MLIV patients are Ashkenazi Jewish, and the two founder mutations, present in 95% of Ashkenazi Jewish patients, result in complete loss of mRNA and protein.¹⁰ MLIV is a rare disease with carrier frequency of 1:100 in Ashkenazi Jewish and 1:10,000 in the general population. Many patients with MLIV remain undiagnosed or are misdiagnosed with cerebral palsy. Thus, bringing awareness of this disease to pediatric ophthalmologists and neurologists is important to improve diagnosis as new therapies are developed.Mucolipin-1 has six transmembrane domains and is permeable to Ca²⁺, Na⁺, K⁺, Fe²⁺, Mn²⁺, and Zn²⁺.^{11, 12, 13} Its channel activity is regulated by both calcium concentration and pH, and mucolipin-1 has been shown to have lipase activity.¹⁴ Previous studies by our group and others showed mucolipin-1 localization to the late endosomes and lysosomes.^{15, 16, 17, 18} The transient receptor potential channel protein mucolipin-1 is required for transport of lipids from the late endosomes-lysosomes to the trans-Golgi compartment,^{19, 20} Ca²⁺-dependent late endosome-lysosome fission-fusion events,²⁰ reformation of lysosomes from endosome-lysosome hybrids^{18, 21} and autolysosomes,^{22, 23} and lysosomal exocytosis.^{24, 25} Mucolipin-1 is strongly expressed in the mouse retina, with the highest mRNA levels in the outer plexiform layer and outer nuclear layer.²⁶The Mcoln1 knockout (KO) mouse model recapitulates the main features of the human disease, and is a good phenotypic platform for investigating MLIV disease mechanisms.^{27, 28, 29, 30} At the ultrastructural level, typical MLIV storage inclusions have been found in the brain during embryonic development.³¹ Histochemical analysis in the young adult (2 months of age) Mcoln1^−/− mice has shown pronounced glial activation, reduced myelination, and no neuronal loss in the cerebrum in the regions most affected by gliosis.²⁸In this study, we used Mcoln1^−/− mice to characterize the consequences of mucolipin-1 loss on retinal morphology, optic nerve myelination, and visual function in the course of the disease. Major manifestations of ophthalmic pathology in Mcoln1^−/− mice were as follows: non-progressive thinning of the photoreceptor layer; profound accumulation of storage inclusions throughout the retina and in the optic nerve, including formation of large axonal spheroids; axonal degeneration in the optic nerve in older mice; hypertrophied lysosomes in photoreceptors and other retinal cells; and reduced visual function.     

Loss of Cxcr4 in Endometriosis Reduces Proliferation and Lesion Number while Increasing Intraepithelial Lymphocyte Infiltration

Hyperactivation of the CXCL12-CXCR4 axis occurs in endometriosis; the therapeutic potential of treatments aimed at global inhibition of the axis was recently reported. Because CXCR4 is predominantly expressed on epithelial cells in the uterus, this study explored the effects of targeted disruption of CXCR4 in endometriosis lesions. Uteri derived from adult female mice homozygous for a floxed allele of CXCR4 and co-expressing Cre recombinase under control of progesterone receptor promoter were sutured onto the peritoneum of cycling host mice expressing the green fluorescent protein. Four weeks after endometriosis induction, significantly lower number of lesions developed in Cxcr4-conditional knockout lesions relative to those in controls (37.5% vs. 68.8%, respectively). In lesions that developed in Cxcr4-knockout, reduced epithelial proliferation was associated with a lower ratio of epithelial to total lesion area compared with controls. Furthermore, while CD3⁺ lymphocytes were largely excluded from the epithelial compartment in control lesions, in Cxcr4-knockout lesions, CD3⁺ lymphocytes infiltrated the Cxcr4-deficient epithelium in the diestrus and proestrus stages. Current data demonstrate that local CXCR4 expression is necessary for proliferation of the epithelial compartment of endometriosis lesions, that its downregulation compromises lesion numbers, and suggest a role for epithelial CXCR4 in lesion immune evasion.

Endometriosis is one of the most common gynecologic diseases in women of reproductive age, with a prevalence rate of approximately 10%.¹ Various theories have been proposed for the origin of endometriosis, including the most widely accepted theory of retrograde menstruation, in which shed endometrial tissue is refluxed through the fallopian tubes and proliferates within the pelvis.^2,3 Because the majority of women have retrograde menstruation, yet only about one in 10 develops endometriosis, it has been proposed that factors promoting survival, invasiveness, and growth of endometrial fragments in the peritoneal cavity play a role in their persistence at ectopic sites in women with endometriosis. Such predisposing factors include somatic mutations in the highly proliferative endometrial epithelium (ie, KRAS, ARID1A⁴), aberrant progenitor/stem cell populations (endometrial or bone marrow (BM) derived5, 6, 7, 8, 9) at ectopic sites, and/or an immune-tolerant microenvironment permissive to proliferation and neoangiogenesis of ectopic endometrial fragments. This immunosuppressive microenvironment is characterized by elevated levels of activated peritoneal macrophages, reduced natural killer cell activity, and abnormally high levels of T-regulatory cells,¹⁰ which suppress immune mechanisms aimed at eliminating implantation of misplaced autologous cells.The chemokine-receptor CXCL12-CXCR4 axis is up-regulated in endometriosis.11, 12, 13, 14 The axis has roles in promoting cell survival, proliferation, chemotaxis, invasion, and angiogenesis. In cancer, hyperactivation of the axis is associated with disease progression and correlates with poor clinical outcome.15, 16, 17, 18, 19 This axis was also proposed to function in immune modulation: CXCL12 binding to CXCR4-expressing intratumoral (epithelial) cells was suggested to be a mechanism mediating cancer evasion of immune surveillance.^20,21 Therapeutic blockade of the axis with the CXCR4 antagonist AMD3100 exhibited antitumor effects, including reduced tumor proliferation and increased apoptosis, both associated with T-cell accumulation within the tumor epithelium.^20,22, 23, 24Endometrial CXCR4 is predominantly expressed on luminal and glandular epithelia, whereas the stroma is the principal source of the ligand CXCL12.^13,25 Stromal-derived CXCL12 exerts its proliferative effect on the epithelium through paracrine interactions with its cognate receptor CXCR4.²⁶ Estradiol stimulates CXCL12 production and progesterone to inhibit this stimulation.^27,28 In vitro, AMD3100 blocked the CXCL12-mediated proliferative effects on epithelial cells.²⁹ Acute treatment of experimental endometriosis in mice with AMD3100 significantly decreases lesion volume and reduces BM cell trafficking to lesions.³⁰ AMD3100 was also shown to reduce recruitment of BM-derived endothelial progenitor cells into lesions and compromise their vascularization.³¹ Based on these studies, whether the inhibitory action of AMD3100 on lesion growth is mediated via local effects (ie, inhibiting lesion-endogenous CXCR4) or systemic effects (ie, inhibiting exogenous CXCR4-expressing cells, which infiltrate lesions with endometriosis induction) was explored. Moreover, in a manner similar to cancer, lesion-derived CXCR4 may have a role in immune evasion.To achieve these goals, endometriosis was induced using uteri derived from 8- to 10- week–old Pgr^Cre/+ Cxcr4^−/− female mice homozygous for a floxed CXCR4 allele and harboring a progesterone receptor promoter–driven Cre recombinase. Endometriosis was induced in syngeneic green fluorescent protein (GFP) transgenic host mice, allowing discrimination of host-derived populations from endometrial cells within uterine explants. A significant reduction in the number of lesions was found in mice harboring Cxcr4-conditional knockout lesions. In lesions that did develop, epithelial thinning was observed concomitant with the appearance of intraepithelial lymphocytes. At the proliferative stage, Ki-67 staining was absent from the epithelium of lesions, suggesting that diminished lesion numbers may be attributed to loss of epithelial proliferation, ultimately undermining lesion integrity.     

VEGFA Activates Erythropoietin Receptor and Enhances VEGFR2-Mediated Pathological Angiogenesis

Clinical and animal studies implicate erythropoietin (EPO) and EPO receptor (EPOR) signaling in angiogenesis. In the eye, EPO is involved in both physiological and pathological angiogenesis in the retina. We hypothesized that EPOR signaling is important in pathological angiogenesis and tested this hypothesis using a rat model of oxygen-induced retinopathy that is representative of human retinopathy of prematurity. We first determined that EPOR expression and activation were increased and that activated EPOR was localized to retinal vascular endothelial cells (ECs) in retinas at postnatal day 18 (p18), when pathological angiogenesis in the form of intravitreal neovascularization occurred. In human retinal microvascular ECs, EPOR was up-regulated and activated by VEGF. Lentiviral-delivered shRNAs that knocked down Müller cell–expressed VEGF in the retinopathy of prematurity model also reduced phosphorylated EPOR (p-EPOR) and VEGFR2 (p-VEGFR2) in retinal ECs. In human retinal microvascular ECs, VEGFR2-activated EPOR caused an interaction between p-EPOR and p-VEGFR2; knockdown of EPOR by siRNA transfection reduced VEGF-induced EC proliferation in association with reduced p-VEGFR2 and p-STAT3; however, inhibition of VEGFR2 activation by siRNA transfection or semaxanib (SU5416) abolished VEGFA-induced proliferation of ECs and phosphorylation of VEGFR2, EPOR, and STAT3. Our results show that VEGFA-induced p-VEGFR2 activates EPOR and causes an interaction between p-EPOR and p-VEGFR2 to enhance VEGFA-induced EC proliferation by exacerbating STAT3 activation, leading to pathological angiogenesis.Retinopathy of prematurity (ROP) is an important cause of vision loss and blindness in infants worldwide.¹ Because of the limited ability to study human preterm infant eyes, models have been established in which newborn animals that normally vascularize their retinas after birth are exposed to oxygen stresses that lead to retinal features similar to human ROP.² Based on such models of oxygen-induced retinopathy (OIR) and on observations in human infants, ROP has been described as having two phases.^3,4 In phase I, infants experience delayed physiological retinal vascular development and sometimes vasoattenuation from high oxygen.⁵ Phase II is characterized by aberrant disordered developmental angiogenesis in the form of vasoproliferative intravitreal neovascularization (IVNV).² Several angiogenic agonists and inhibitors have been recognized as potentially involved in human ROP. Of these, the most studied is vascular endothelial growth factor A (VEGFA).^6–9Besides being involved in human pathological angiogenic eye disease,¹⁰ VEGFA is also important in retinal vascular development.^11,12 Inhibition of the bioactivity of VEGFA in preterm infants with severe ROP reduced the IVNV of phase II,¹³ but reports of persistent avascular retina and recurrent pathological angiogenesis raised concern.¹⁴ Furthermore, neutralizing VEGFA with an antibody similar to that used in human preterm infants with severe ROP initially reduced IVNV in the rat ROP model, but caused recurrent pathological angiogenesis in association with up-regulation of several angiogenic agonists, including erythropoietin (EPO).¹⁵EPO is known mainly for hematopoiesis, and it has been used to treat anemia.¹⁶ However, a growing body of evidence indicates that EPO has other biological effects, including neuroprotective,^17–19 antiapoptotic,^19,20 antioxidative,^20,21 and angiogenic^22–24 properties. Evidence supporting the role of EPO in angiogenesis comes from clinical and animal studies. In clinical studies, proliferative diabetic retinopathy and severe ROP have been associated with increased EPO. In proliferative diabetic retinopathy, vitreous EPO was increased,²⁵ and a promoter polymorphism in the EPO gene resulting in increased production of EPO was associated with severe diabetic retinopathy in a largely European-American population.²⁶ In ROP, greater risk of severe ROP was associated with EPO treatment for anemia of prematurity.^27,28 In a mouse OIR model, hyperoxia down-regulated EPO expression in the retina and decreased vascular stability in association with vaso-obliterated retina,²⁹ and, after relative hypoxia, retinal EPO was increased and contributed to IVNV.³⁰ EPO was also identified as a target in OIR in a study using a transgenic mouse in which hypoxia inducible factor 2α (HIF-2α; EPAS1, alias HLF) was knocked down,³¹ and EPO synergistically increased VEGFA-induced human retinal microvascular endothelial cell (hRMVEC) proliferation.¹⁵ However, EPO also promoted physiological retinal vascularization in a rat OIR model.³² Thus, the evidence is mixed, in that EPO has been associated with both physiological and pathological retinal angiogenesis. EPO is now being considered as a neuroprotective agent to promote cognitive development in preterm infants.³³ Greater understanding is needed regarding EPO and EPO receptor (EPOR) signaling in ROP and developmental angiogenesis.In the present study, we used a rat ROP model³⁴ in which VEGFA is overexpressed by postnatal day 12 (p12) and causes IVNV at p18.³⁵ Our research group previously found that the VEGFA signal is detected in Müller cells and developed a method using a lentiviral vector that targets Müller cells and knocks down VEGFA in vivo, thereby inhibiting IVNV without interfering with pup growth or serum VEGFA.³⁶ The present investigation of the role of EPOR adapted lentiviral vector rat model. In phase I, EPOR activation was lower than in phase II, when VEGFA expression and VEGFR2 expression and activation were increased.³⁷ In hRMVECs, VEGFA up-regulated and activated EPOR and (through crosstalk between activated VEGFR2 and EPOR) increased the activation of STAT3 to enhance angiogenesis. Our present findings support the hypothesis that VEGFA activates and causes an interaction between EPOR and VEGFR2 to contribute to pathological angiogenesis.