Tuberin Regulates E-Cadherin Localization: Implications in Epithelial-Mesenchymal Transition

The tuberous sclerosis complex 2 (TSC2) gene encodes the protein tuberin, which functions as a key negative regulator of both mammalian target of rapamycin (mTOR) C1-dependent cell growth and proliferation. Loss-of-function mutations of TSC2 result in mTORC1 hyperactivity and predispose individuals to both tuberous sclerosis and lymphangioleiomyomatosis. These overlapping diseases have in common the abnormal proliferation of smooth muscle-like cells. Although the origin of these cells is unknown, accumulating evidence suggests that a metastatic mechanism may be involved, but the means by which the mTOR pathway contributes to this disease process remain poorly understood. In this study, we show that tuberin regulates the localization of E-cadherin via an Akt/mTORC1/CLIP170-dependent, rapamycin-sensitive pathway. Consequently, Tsc2(−/−) epithelial cells display a loss of plasma membrane E-cadherin that leads to reduced cell-cell adhesion. Under confluent conditions, these cells detach, grow in suspension, and undergo epithelial-mesenchymal transition (EMT) that is marked by reduced expression levels of both E-cadherin and occludin and increased expression levels of both Snail and smooth muscle actin. Functionally, the Tsc2(−/−) cells demonstrate anchorage-independent growth, cell scattering, and anoikis resistance. Human renal angiomyolipomas and lymphangioleiomyomatosis also express markers of EMT and exhibit an invasive phenotype that can be interpreted as consistent with EMT. Together, these results suggest a novel relationship between TSC2/mTORC1 and the E-cadherin pathways and implicate EMT in the pathogenesis of tuberous sclerosis complex-related diseases.Mutation of the TSC2 gene gives rise to the autosomal dominant disorder, tuberous sclerosis complex (TSC), that is characterized by “hamartomas” in the brain, kidney, skin, heart, and lung.¹ Genetic and biochemical analyses have highlighted the role of the TSC2 protein, tuberin, in concert with its interacting partner, TSC1 (hamartin), in negatively regulating mammalian target of rapamycin (mTOR) C1 by promoting the hydrolysis of Rheb-GTP.² Multiple factors including growth factors, energy, and oxygen availability converge on the TSC1/TSC2 complex to modulate mTORC1 activity.³ The best characterized function of mTORC1 is the promotion of protein synthesis through its downstream targets, p70S6K and 4E-BP1. In turn, p70S6K mediates phosphorylation of IRS-1 to inhibit phosphatidylinositol 3-kinase/AKT signaling in a negative feedback mechanism.⁴ Consequently, the loss of TSC1 or TSC2 leads to an “overgrowth” phenotype with increased cell size and proliferation, characteristic of the hamartomas seen in tuberous sclerosis. However, many of the clinical and pathological features of TSC remain unexplained by our current understanding of the function of these genes.One such example is the lymphangioleiomyomatosis (LAM) that occurs in ∼40% of females diagnosed with TSC.⁵ The sporadic form of LAM is also associated with mutation of the TSC2 gene.⁶ LAM is a unique disease that affects women of childbearing age and is characterized by the infiltration of smooth muscle-like cells in the lung interstitium, which eventually leads to the progressive loss of pulmonary function and cystic destruction of the lung.⁷ Although LAM is not exclusive to the lungs and can involve the axial lymphatic system and other organs, mortality due to respiratory failure takes place within 8 to 15 years after diagnosis.⁸ LAM and angiomyolipoma (AML) are classified as perivascular epithelioid cell neoplasms), that is, defined as “mesenchymal tumors composed of histologically and immunohistochemically distinctive perivascular epithelioid cells.”⁹ These tumors are characterized histologically by their epithelioid appearance and their physical relationship to blood vessels.⁹ The abnormal cells display a distinct immunophenotype that includes the expression of melanocytic (eg, gp100) and smooth muscle markers (eg, smooth muscle actin) but not epithelial antigens. Because perivascular epithelioid cells have no normal anatomical counterpart, the origin of these tumors remains elusive.One current theory suggests that pulmonary LAM is the result of a metastatic process in which certain precursor cells migrate to the lung and invade the parenchyma.¹⁰ Indeed, primary LAM cells have been shown to be invasive in vitro,¹¹ and these cells have been identified in body fluids, including blood and urine, suggesting that LAM cells are capable of detaching from their primary sites and entering the circulation.¹² Further evidence in support of this hypothesis comes from the observations that pulmonary recurrences of LAM after lung transplant contain cells that originated from the organ recipients,^13,14 and common patterns of TSC2 mutation have been identified in LAM and lymph node disease from the same individual.¹⁵ Finally, the unique immunophenotype in LAM does not reflect an epithelial nor mesenchymal origin but rather a mixture of “epithelioid” and “spindle” cells that is suggestive of a variable differentiation pattern.¹⁶ Nonetheless, both populations of cells are believed to be clonally derived. The spindle cells are reported to be more proliferative and express smooth muscle actin, whereas the epithelioid cells express the melanocytic markers (eg, HMB-45) and are less mitotically active. Collectively, the observed behavior of LAM cells with respect to their infiltrative growth pattern, metastatic potential, and altered cell differentiation is reminiscent of cells undergoing epithelial-mesenchymal transition (EMT).^17,18 Here, we propose that LAM may be a manifestation of EMT and show that human AML and LAM do indeed express markers of EMT.One of the critical steps driving EMT is the repression of E-cadherin, resulting in loss of cell-cell adhesion. E-cadherin is expressed in most epithelial cells in which adherens junctions are formed to create the multicellular organization important for the formation and maintenance of bodily compartments. Structural studies highlight the essential role of calcium in the progressive cis-dimerization of the cadherin ectodomain, leading to the formation of a trans-dimer “zipper” between multiple cis-dimers to form cell adhesion.¹⁹ In this study, we provide evidence that E-cadherin membrane localization is regulated by the Akt/mTORC1 pathway such that the loss of TSC2 leads to significant reduction in membrane E-cadherin. Consequently, cells deficient in Tsc2 are less adhesive and more prone to detach and to undergo EMT. Our findings highlight a novel functional link between tuberin and E-cadherin activity that may contribute to the pathogenesis of TSC and related disorders.     

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.     

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.     

CXCL9 Is Important for Recruiting Immune T?Cells into the Brain and Inducing an Accumulation of the T Cells to the Areas of Tachyzoite Proliferation to Prevent Reactivation of Chronic Cerebral Infection with Toxoplasma gondii

T cells are required to maintain the latency of chronic infection with Toxoplasma gondii in the brain. Here, we examined the role of non–glutamic acid-leucine-arginine CXC chemokine CXCL9 for T-cell recruitment to prevent reactivation of infection with T. gondii. Severe combined immunodeficient (SCID) mice were infected and treated with sulfadiazine to establish a chronic infection. Immune T cells from infected wild-type mice were transferred into the SCID mice in combination with treatment with anti-CXCL9 or control sera. Three days later, sulfadiazine was discontinued to initiate reactivation of infection. Numbers of CD4⁺ and CD8⁺ T cells isolated from the brains were markedly less in mice treated with anti-CXCL9 serum than in mice treated with control serum at 3 days after sulfadiazine discontinuation. Amounts of tachyzoite (acute stage form of T. gondii)-specific SAG1 mRNA and numbers of foci associated with tachyzoites were significantly greater in the former than the latter at 5 days after sulfadiazine discontinuation. An accumulation of CD3⁺ T cells into the areas of tachyzoite growth was significantly less frequent in the SCID mice treated with anti-CXCL9 serum than in mice treated with control serum. These results indicate that CXCL9 is crucial for recruiting immune T cells into the brain and inducing an accumulation of the T cells into the areas where tachyzoites proliferate to prevent reactivation of chronic T. gondii infection.Toxoplasma gondii is an obligate intracellular parasite in humans and animals. Interferon (IFN)-γ–mediated immune responses^{1, 2} and, to a lesser degree, humoral immunity^{3, 4, 5} control the tachyzoite growth during the acute stage of infection, but the parasite establishes a chronic infection by forming cysts preferentially in the brain. Chronic infection with T. gondii is ubiquitous in humans, and 500 million to 2 billion people worldwide are estimated to be chronically infected with this parasite.^{6, 7} This chronic infection can be reactivated and develop life-threatening toxoplasmic encephalitis (TE) in immunocompromised persons such as those with AIDS, neoplastic diseases, and organ transplants.^{8, 9} This fact clearly indicates an importance of the protective immunity to maintain the latency of chronic infection with T. gondii in the brain. However, the mechanisms by which the immune responses prevent reactivation of the chronic infection are not well understood.Although T. gondii has three major genotypes (types I, II, and III), type II is predominant in the strains isolated from patients with TE in North America and Europe.^{10, 11, 12} Therefore, for investigating the mechanisms by which the immune system maintains the latency of chronic T. gondii infection and prevents TE, animals that establish a latent, chronic infection with type II parasite in their brains provide an excellent model. BALB/c mice are one of those animals.^{13, 14} IFN-γ is essential to maintain the latency of chronic cerebral infection with T. gondii.^{15, 16} This cytokine can activate microglia^{17, 18} and astrocytes^{19, 20, 21} to prevent tachyzoite proliferation. In addition, IFN-γ plays an important role in regulating recruitment of immune T cells into the brain of BALB/c mice during both the acute and chronic stages of infection.^{22, 23} Induction of vascular cell adhesion molecule 1 (VCAM-1) expression on the cerebral vessels during the chronic infection is largely mediated by IFN-γ,²² and the binding of α4β1 integrin expressed on the surface of T cells to VCAM-1 expressed on the cerebrovascular endothelial cells is important for inducing prompt recruitment of immune T cells into their brains during the early stage of reactivation of chronic T. gondii infection to prevent TE.²⁴Chemokines, in addition to adhesion molecules, are crucial for T-cell entry into various organs.^{25, 26} In BALB/c mice chronically infected with T. gondii, CXCL9, CXCL10, and CCL5 are the chemokines predominantly expressed in their brains.^{27, 28} In the present study, we examined the role of IFN-γ in cerebral expression of these three chemokines during reactivation of the chronic infection in BALB/c-background immunodeficient mice, and found that the CXCL9 expression requires IFN-γ. On the basis of this observation, we examined the role of CXCL9 in recruiting immune T cells into the brain for maintaining the latency of chronic infection with T. gondii with the use of a model of reactivation of the infection in severe combined immunodeficient (SCID) mice with adoptive transfer of immune T cells from infected wild-type animals. By applying anti-CXCL9 antiserum in this animal model, the present study revealed that CXCL9 is crucial for recruiting immune T cells into the brain and for inducing an accumulation of the T cells around the areas associated with tachyzoites to prevent reactivation of cerebral infection with T. gondii.     

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.     

p120-Catenin Expressed in Alveolar Type II Cells Is Essential for the Regulation of Lung Innate Immune Response

The integrity of the lung alveolar epithelial barrier is required for the gas exchange and is important for immune regulation. Alveolar epithelial barrier is composed of flat type I cells, which make up approximately 95% of the gas-exchange surface, and cuboidal type II cells, which secrete surfactants and modulate lung immunity. p120-catenin (p120; gene symbol CTNND1) is an important component of adherens junctions of epithelial cells; however, its function in lung alveolar epithelial barrier has not been addressed in genetic models. Here, we created an inducible type II cell–specific p120-knockout mouse (p120EKO). The mutant lungs showed chronic inflammation, and the alveolar epithelial barrier was leaky to ¹²⁵I-albumin tracer compared to wild type. The mutant lungs also demonstrated marked infiltration of inflammatory cells and activation of NF-κB. Intracellular adhesion molecule 1, Toll-like receptor 4, and macrophage inflammatory protein 2 were all up-regulated. p120EKO lungs showed increased expression of the surfactant proteins Sp-B, Sp-C, and Sp-D, and displayed severe inflammation after pneumonia caused by Pseudomonas aeruginosa compared with wild type. In p120-deficient type II cell monolayers, we observed reduced transepithelial resistance compared to control, consistent with formation of defective adherens junctions. Thus, although type II cells constitute only 5% of the alveolar surface area, p120 expressed in these cells plays a critical role in regulating the innate immunity of the entire lung.Lungs are constantly exposed to pathogens; therefore, a highly restrictive alveolar epithelial barrier and finely tuned host defense mechanisms are indispensable for their protection.^1,2 Unchecked inflammation is linked to various acute and chronic diseases, including edema, acute respiratory distress syndrome, and fibrosis.^3,4 Although it is abundantly clear that the alveolar epithelial barrier regulates the transport of gases, liquid, and ions,^5,6 the role of the barrier in the regulation of the innate immune function of lungs remains poorly understood.The restrictiveness of the alveolar epithelial barrier is dependent on a series of interacting proteins comprising the adherens junctions (AJs) and tight junctions (TJs).^7,8 The core of the epithelial AJs is composed of E-cadherin, which links cells to one another in the monolayer.⁹ The cytoplasmic domain of E-cadherin associates with α-catenin, β-catenin, and p120-catenin (p120, official name catenin delta 1; CTNND1).⁹ The α- and β-catenins can recruit proteins that link E-cadherin to the actin cytoskeleton,⁹ and together, these interactions maintain the tension landscape in the epithelial monolayer.¹⁰ β-Catenin also plays an essential role in the Wnt signaling pathway and thereby contributes to cell proliferation and differentiation.¹¹ However, p120 has received comparatively less attention, although recent studies have shown that p120 has important functions in regulating cadherin stability and turnover¹² and innate immunity.¹³Here, we focused on the role of p120 expressed in alveolar epithelial type II cells in regulating the innate immune function of lungs. Although alveolar type II cells cover only 5% of the alveolar surface area, these cells are metabolically active.¹⁴ They produce surfactants, serve as facultative progenitor cells to repair alveolar injury, and regulate innate immune function of the lung.¹⁴ These cells express Toll-like receptors (TLRs) and tumor necrosis factor receptors.¹⁵ Interactions with pathogens or endotoxins activate these receptors to initiate NF-κB signaling to produce tumor necrosis factor,¹⁶ IL-1 and IL-6,¹⁶ regulated on activation normal T cell expressed and secreted,¹⁷ and chemokine C-X-C motif ligand 1.¹⁸ These factors play key roles in recruiting inflammatory cells.^19–21 Alveolar type II cells also secrete the surfactant proteins (Sp)-A, -B, -C, and -D,²² which regulate innate and adaptive immunity by binding to antigen through interactions with surface receptors on inflammatory cell membranes.²³ Here, we studied the function of p120 through disrupting the p120 gene in alveolar type II cells in mice using the rtTA/TetO system coupled with a type II cell–specific SPC promoter. In these mice, we observed unchecked chronic lung inflammation associated with increased NF-κB activity and a persistently leaky alveolar epithelial barrier. These results provide the first genetic evidence that p120 in type II cells is a central regulator of innate immunity of lungs.     

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.     

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.     

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.     

Endometrial Cancer Side-Population Cells Show Prominent Migration and Have a Potential to Differentiate into the Mesenchymal Cell Lineage

Cancer stem-like cell subpopulations, referred to as “side-population” (SP) cells, have been identified in several tumors based on their ability to efflux the fluorescent dye Hoechst 33342. Although SP cells have been identified in the normal human endometrium and endometrial cancer, little is known about their characteristics. In this study, we isolated and characterized the SP cells in human endometrial cancer cells and in rat endometrial cells expressing oncogenic human K-Ras protein. These SP cells showed i) reduction in the expression levels of differentiation markers; ii) long-term proliferative capacity of the cell cultures; iii) self-renewal capacity in vitro; iv) enhancement of migration, lamellipodia, and, uropodia formation; and v) enhanced tumorigenicity. In nude mice, SP cells formed large, invasive tumors, which were composed of both tumor cells and stromal-like cells with enriched extracellular matrix. The expression levels of vimentin, α-smooth muscle actin, and collagen III were enhanced in SP tumors compared with the levels in non-SP tumors. In addition, analysis of microdissected samples and fluorescence in situ hybridization of Hec1-SP-tumors showed that the stromal-like cells with enriched extracellular matrix contained human DNA, confirming that the stromal-like cells were derived from the inoculated cells. Moreober, in a Matrigel assay, SP cells differentiated into α-smooth muscle actin-expressing cells. These findings demonstrate that SP cells have cancer stem-like cell features, including the potential to differentiate into the mesenchymal cell lineage.Recently, adult stem cells have been identified in several mature tissues, such as the adult intestine,¹ skin,² muscle,³ blood,⁴ and the nervous system^5–7 A stem cell is an undifferentiated cell that is defined by its ability to both self-renew and to produce mature progeny cells.⁸ Stem cells are classified based on their developmental potential as totipotent, pluripotent, oligopotent, and unipotent. Adult somatic stem cells were originally thought to be tissue specific and only able to give rise to progeny cells corresponding to their tissue of origin. Recent studies, however, have shown that adult mammalian stem cells are able to differentiate across tissue lineage boundaries,^9,10 although this “plasticity” of adult somatic stem cells remains controversial.Stem cell subpopulations (“side-population” (SP) cells) have been identified in many mammals, including humans, based on the ability of these cells to efflux the fluorescent dye Hoechst 33342.¹¹ Recent evidence suggests that the SP phenotype is associated with a high expression level of the ATP-binding cassette transporter protein ABCG2/Bcrp1.¹² Most recently, established malignant cell lines, which have been maintained for many years in culture, have also been shown to contain SP cells as a minor subpopulation.¹³The human endometrium is a highly dynamic tissue undergoing cycles of growth, differentiation, shedding, and regeneration throughout the reproductive life of women. Endometrial adult stem/progenitor cells are likely responsible for endometrial regeneration.¹⁴ Rare populations of human endometrial epithelial and stromal colony-forming cells¹⁵ and SP cells^16,17 have been identified. Although coexpression of CD146 and PDGFRβ isolates a population of mesenchymal stem like cells from human endometrium,¹⁸ specific stem cell markers of endometrium remain unclear. Recently, Gotte et al¹⁹ demonstrated that the adult stem cell marker Musashi-1 was coexpressed with Notch-1 in a subpopulation of endometrial cells. Furthermore, they showed that telomerase and Musashi-1-expressing cells were significantly increased in proliferative endometrium, endometriosis, and endometrial carcinoma tissue, compared with secretary endometrium, suggesting the concept of a stem cell origin of endometriosis and endometrial carcinoma.Recent evidence suggests that cancer stem-like cells exist in several malignant tumors, such as leukemia^20,21 breast cancer,²² and brain tumors,²³ and that these stem cells express surface markers similar to those expressed by normal stem cells in each tissue.^20,24Development of endometrial carcinoma is associated with a variety of genetic alterations. For example, increased expression and activity of telomerase^25,26 and frequent dysregulation of signaling pathways have been observed in endometrial carcinoma. Some of these pathways are important determinants of stem cell activity (Wnt-β-catenin and PTEN).^27–29 These suggest a stem cell contribution to endometrial carcinoma development.Recently, we isolated SP cells from the human endometrium. These SP cells showed long-term proliferating capacity in cultures and produced both gland and stromal-like cells. Additionally, they were able to function as progenitor cells.¹⁶ In this study, we isolated and characterized SP cells from human endometrial cancer cells and from rat endometrial cells expressing oncogenic [¹²Val] human K-Ras protein and demonstrated their cancer stem-like cell phenotypes.     

Immunogenicity of Decellularized Porcine Liver for Bioengineered Hepatic Tissue

Liver disease affects millions of patients each year. The field of regenerative medicine promises alternative therapeutic approaches, including the potential to bioengineer replacement hepatic tissue. One approach combines cells with acellular scaffolds derived from animal tissue. The goal of this study was to scale up our rodent liver decellularization method to livers of a clinically relevant size. Porcine livers were cannulated via the hepatic artery, then perfused with PBS, followed by successive Triton X-100 and SDS solutions in saline buffer. After several days of rinsing, decellularized liver samples were histologically analyzed. In addition, biopsy specimens of decellularized scaffolds were seeded with hepatoblastoma cells for cytotoxicity testing or implanted s.c. into rodents to investigate scaffold immunogenicity. Histological staining confirmed cellular clearance from pig livers, with removal of nuclei and cytoskeletal components and widespread preservation of structural extracellular molecules. Scanning electron microscopy confirmed preservation of an intact liver capsule, a porous acellular lattice structure with intact vessels and striated basement membrane. Liver scaffolds supported cells over 21 days, and no increased immune response was seen with either allogeneic (rat-into-rat) or xenogeneic (pig-into-rat) transplants over 28 days, compared with sham–operated on controls. These studies demonstrate that successful decellularization of the porcine liver could be achieved with protocols developed for rat livers, yielding nonimmunogenic scaffolds for future hepatic bioengineering studies.Within the United States alone, tens of thousands of patients are awaiting a liver transplant, with only a few thousand donor organs available annually.¹ This widening mismatch has led physicians and researchers to pursue alternative therapies for chronic liver disease, including in situ cell-based therapies or xenotransplantation of organs.^2–4 The field of regenerative medicine offers another approach, in which elements of both would be combined for the bioengineering of neo-organs for transplantation.^5,6The concept of whole liver tissue engineering aims to combine patient-specific autologous hepatocytes or hepatic progenitor cells and a carrying platform, or scaffold, to allow for three-dimensional tissue growth and permit the complex cellularity of hepatic tissue. Use of decellularized organ matrices preserves the natural extracellular matrix (ECM) proteins and growth factors that guide cell attachment and proliferation in an organ-specific manner.⁷ Proper processing of the matrix scaffolds removes all cytotoxic chemicals from the decellularization process and performs complete degradation of donor nucleic acids to prevent an adverse host immune response.⁸ These bioengineered livers have the ultimate potential to surpass the current allograft gold standard.The process begins by removing the native cellular components from a donor tissue using detergents and enzymes and leaving behind an ECM scaffold with preserved vasculature and essential biological factors. The concept has been applied to many tissues, including the heart,^9,10 lungs,^11–14 bladder,¹⁵ blood vessels,^16,17 muscle,¹⁸ intestines,^19,20 trachea,^21–23 kidney,^7,24,25 and liver.^26–30 Each detergent, enzyme, washing buffer, and sterilization technique used to decellularize a tissue can have a direct influence on the host remodeling response and functional outcome.³¹ In a previous study, decellularized matrix scaffolds were immunologically favorable up until cells were added to the scaffolding material, where proinflammatory macrophages were activated.³² To evaluate whether a decellularized tissue represents a viable scaffold option, the generated matrices need to be implanted and evaluated over time, without cells, to allow a host’s immune cells to infiltrate and respond to the material.³³ The initial response can begin as early as 2 days and last for months.³⁴ During that time, the environment from both the host itself and the degrading matrix material can influence the phenotype of the host immune cells switching between activation states that will determine the future clinical viability of the biological matrix material.^35–37 Triggering of proinflammatory macrophage activation results in the release of cytokines, growth factors, proteolytic enzymes, and reactive oxygen and nitrogen intermediates that will greatly inhibit the integration of the biomaterial with the host tissue.³⁸ Studies on the immunogenicity of decellularized whole tissue are limited, and a key criterion of transplantation viability will be evaluating the activation of host macrophages toward the classically proinflammatory phenotype (M1) or the regenerative and repair phenotype (M2).The objectives of the current study were to generate a decellularized porcine liver by scaling up our previously established rodent perfusion protocol,²⁸ characterize the resultant scaffold, and compare the in vivo immunological response of a rodent host between allograft and xenograft decellularized liver matrices. We hypothesized that both tissues would elicit a similar host response as the result of the high levels of preservation the ECM protein structures share between species.³⁹ The generation of large-scale hepatic tissue platforms, and an understanding of the inherent immune response by a host species, will be vital in producing implantable bioengineered livers.     

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.     

Exposure to Microbial Metabolite Butyrate Prolongs the Survival Time and Changes the Growth Pattern of Human Papillomavirus 16 E6/E7-Immortalized Keratinocytes in Vivo

Human papillomavirus (HPV) is a ubiquitous human pathogen that can be cleared by host immunity. Nonetheless, a small percentage of the patients develop persistent infection with oncogenic HPV, which poses an increased risk of developing HPV-associated malignancy. Although cell-mediated immunity is a known systemic factor, local factors that influence persistent HPV infection have not been fully investigated. HPV-related head/neck cancers have a strong site preference for the oropharynx, suggesting the existence of unique local factors that promote HPV-induced oncogenesis. The human oropharynx often harbors anaerobic bacteria that produce a variety of byproducts, including butyrate. Because butyrate is a potent epigenetic modulator, it could be an environmental factor influencing the development of HPV-positive oropharyngeal malignancy. In this study, we showed that butyrate treatment changed the property of HPV16 E6/E7-immortalized keratinocytes. In vitro, the treatment increased the cells'' migration ability, slowed the growth, and increased the genotoxic resistance. When implanted in the syngeneic mice, the treated keratinocytes survived longer and exhibited a different growth pattern. The survival advantage obtained after butyrate exposure may increase the susceptibility of HPV-infected oropharyngeal keratinocytes to further malignant transformation. These results suggest that fermentation products of tonsillar bacteria may play an important role in the long-term persistence of high-risk HPV infection, which is a critical risk factor for developing HPV-positive oropharyngeal malignancy.

Human papillomavirus (HPV) is a small nonenveloped circular double-stranded DNA virus that infects keratinocytes. Among the five genera identified so far, α-papillomavirus is the most commonly studied group because of its association with cancer/precancerous lesions,¹ and is subdivided into low-risk (eg, HPV6 and HPV11) and high-risk (eg, HPV16 and HPV18) genotypes. High-risk HPV encodes two major oncoproteins, E6 and E7, that play a pivotal role in driving the infected keratinocytes toward malignancy. The E6 oncoprotein inactivates the tumor suppressor and cell cycle checkpoint protein TP53, whereas the E7 oncoprotein inactivates the retinoblastoma (RB1) tumor suppressor protein.^2,3 Together, E6/E7 oncoproteins target diverse cellular pathways involving cell cycle, apoptosis, and cell polarity, resulting in cell cycle deregulation and genome instability.^4,5 Knockdown of E6/E7 induces cell senescence or apoptosis, highlighting their crucial roles in the persistence of HPV-mediated cancers.^6,7 Infection with high-risk HPV is responsible for ≈5% of all human cancers and accounts for 70% of oropharyngeal malignancies.⁸HPV infection has caused an epidemic increase in the incidence of oropharyngeal cancers worldwide.⁹ HPV-related head and neck squamous cell carcinomas (SCCs) occur almost exclusively in the oropharynx.^10,11 HPV-positive oropharyngeal SCC (OPSCC) often exhibits a high-grade morphology, nonkeratinized and basaloid, deviating from the conventional well-differentiated, keratinized SCC in the oral cavity.¹² HPV-positive OPSCC appears to be molecularly and pathologically distinctive from its HPV-negative counterpart. Clinically, HPV-positive OPSCC has a unique pattern of metastasis, a longer disease-free interval, and a better prognosis.¹³ A new cancer staging system for HPV-positive OPSCC was established to guide treatment,¹⁴ partly because de-escalation of treatment does not compromise the cure rate but decreases morbidity caused by adverse effects of the therapy. However, treatment de-escalation for HPV-positive OPSCC has been recently discouraged after the publication of RTOG 1016 and De-ESCALaTE studies, both showing that outcomes of HPV-positive OPSCC patients strongly depend on the type of treatment received.^15,16HPV-related premalignant progression is traditionally studied on uterine cervical premalignant lesions. The derived cell lines have been the major tools for researchers to understand the biological process of HPV-associated malignant transformation.¹⁷ After prolonged passaging, the cell lines are able to undergo phenotypic progression from low-grade through high-grade dysplasia to invasiveness when differentiating into stratified squamous epithelium in organotypic raft epithelial tissue cultures.^18,19 Unlike cervical cancers, the process involving HPV-positive oropharyngeal malignancy is less well understood, and at least two major issues remain to be elucidated. First, premalignant lesions (dysplasia) have rarely, if ever, been identified in clinical oropharyngeal specimens in the absence of an invasive component.²⁰ Because premalignant lesions are present in HPV-related SCCs involving other anatomic sites, including the adjacent oral cavity,²¹ their absence in the oropharynx remains an enigma. Second, HPV-related head and neck cancers have a strong site preference for the oropharynx.^10,22The unusually high incidence of HPV-positive SCC in the oropharynx suggests the existence of unique local factors that promote HPV-induced oncogenesis. The oropharynx contains palatine tonsils and accessory lymphoid tissues of the Waldeyer ring that form deep crypts lined by patches of nonkeratinized stratified squamous epithelium and reticulated spongiotic epithelium with a gapped, noncontinuous basement membrane.²³ The crypt epithelium is constantly infiltrated by lymphoid cells and forms a loose network abutting the subjacent lymphoid tissue. The deep crypts provide a low-oxygen habitat for anaerobic bacteria,^24,25 which produce a variety of fermentation products, including butyrate, which contributes to halitosis.²⁶ Butyrate is a histone deacetylase (HDAC) inhibitor and has been shown to function as an epigenetic modulator to condition intestinal immunity and improve the health of intestinal epithelium.^27,28 However, butyrate has both protumor and anti-tumor properties that have been observed on cultured HPV-immortalized epithelial cells.^29,30Although HPV is a ubiquitous human pathogen, host immunity is capable of clearing the infection in most cases, including those caused by the high-risk group. However, a small percentage of the patients develop persistent infection, which is associated with an increased risk of cancer development.31, 32, 33 Although humans are the natural host of HPV, various studies have shown that HPV16 oncoproteins can target mouse TP53 and RB1 pathways similar to those in the human cells.³⁴ Therefore, mice provide a valuable animal model to investigate HPV precancer-host or cancer-host interaction.The current study showed that butyrate treatment changed the in vitro and in vivo properties of HPV16 E6/E7-expressing keratinocytes. The treatment prolonged the survival time and changed the growth pattern of the cells after injection into syngeneic, immunocompetent mice. The results suggest a link between the byproducts produced by tonsillar bacteria and chronic HPV infection.     

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.     

Cytomegalovirus Impairs Cytotrophoblast-Induced Lymphangiogenesis and Vascular Remodeling in an in Vivo Human Placentation Model

We investigated human cytomegalovirus pathogenesis by comparing infection with the low-passage, endotheliotropic strain VR1814 and the attenuated laboratory strain AD169 in human placental villi as explants in vitro and xenografts transplanted into kidney capsules of SCID mice (ie, mice with severe combined immunodeficiency). In this in vivo human placentation model, human cytotrophoblasts invade the renal parenchyma, remodel resident arteries, and induce a robust lymphangiogenic response. VR1814 replicated in villous and cell column cytotrophoblasts and reduced formation of anchoring villi in vitro. In xenografts, infected cytotrophoblasts had a severely diminished capacity to invade and remodel resident arteries. Infiltrating lymphatic endothelial cells proliferated, aggregated, and failed to form lymphatic vessels. In contrast, AD169 grew poorly in cytotrophoblasts in explants, and anchoring villi formed normally in vitro. Likewise, viral replication was impaired in xenografts, and cytotrophoblasts retained invasive capacity, but some partially remodeled blood vessels incorporated lymphatic endothelial cells and were permeable to blood. The expression of both vascular endothelial growth factor (VEGF)-C and basic fibroblast growth factor increased in VR1814-infected explants, whereas VEGF-A and soluble VEGF receptor-3 increased in those infected with AD169. Our results suggest that viral replication and paracrine factors could undermine vascular remodeling and cytotrophoblast-induced lymphangiogenesis, contributing to bleeding, hypoxia, and edema in pregnancies complicated by congenital human cytomegalovirus infection.Human cytomegalovirus (HCMV) is the leading cause of congenital viral infection, with an incidence in the United States of approximately 1% to 3% of live births.¹ Primary maternal HCMV infection during gestation poses a 40% to 50% risk of intrauterine transmission, whereas recurrent infection in seropositive mothers rarely causes disease.^2,3 Symptomatic infants (25%) have intrauterine growth restriction (IUGR) and permanent birth defects, including neurological deficiencies, retinopathy, and sensorineuronal deafness.^4–6 Congenital disease is more severe when primary maternal infection occurs in the first trimester.⁷ IUGR and spontaneous abortion in the absence of fetal HCMV infection can result from placental pathology.^8–10 Placentas infected in early gestation show long-standing damage and fibrosis at the uterine-placental interface, which impairs critical functions and results in a hypoxic intrauterine environment.^10–15 Despite the prevalence and the medical and societal impact of congenital HCMV infection, the mechanisms of virus replication, pathogenesis, and transplacental transmission are still unresolved because of the complex nature of placental development and extreme species specificity of HCMV, which replicates only in human tissues.Differentiating/invading cytotrophoblasts switch to an endothelial phenotype in a process that is similar to vasculogenesis.¹⁶ The cells up-regulate novel adhesion molecules and proteinases that enable their attachment to and invasion of the uterus. Interstitial invasion requires down-regulation of integrins characteristic of epithelial cells and novel expression of the integrins α1β1, α5β1, and αvβ3.¹⁷ Endovascular cytotrophoblasts that remodel uterine blood vessels transform their adhesion receptor phenotype to resemble that of endothelial cells, expressing vascular-endothelial cadherin, platelet-endothelial adhesion molecule-1, and vascular endothelial adhesion molecule-1.^16,18 Like endothelial cells, cytotrophoblasts express substances that influence vasculogenesis and angiogenesis, including the vascular endothelial growth factor (VEGF) family ligands VEGF-A and VEGF-C and receptors VEGFR-1 [fms-like tyrosine kinase 1 (Flt-1)] and VEGFR-3.^19–21 Expression of these molecules changes as the cells differentiate/invade, and they regulate cytotrophoblast survival in the remodeled uterine vasculature. Finally, as hemiallogeneic embryonic/fetal cells, invasive cytotrophoblasts must avoid maternal immune responses. Their expression of the nonclassical major histocompatibility complex (MHC) class I molecules HLA-G^22,23 and HLA-C, which have limited polymorphisms,^24,25 contributes to their lack of immunogenicity.Previous studies led to a rudimentary understanding of HCMV infection of the human placenta and identified several molecular mechanisms that impair functions of differentiating/invading cytotrophoblasts. HCMV infection dysregulates the expression of key integrins required for cell invasiveness,^26,27 reduces the expression of matrix metalloproteinase-9,²⁷ and down-regulates cell-cell and cell-matrix adhesion molecules,²⁸ including those required for pseudovasculogenesis¹⁶ and vascular remodeling.¹⁸ The immunosuppressive viral cytokine cmv IL-10 further reduces cytotrophoblast invasion through paracrine effects that increase IL-10 expression.^27,28 Peroxisome proliferator-activated receptor γ activation by infection also compromises cytotrophoblast functions.^29,30 In chorionic villi, the neonatal Fc receptor for IgG, expressed in syncytiotrophoblasts that contact maternal blood, transcytoses circulating maternal antibodies.^31–33 In conjunction with neutralizing titers, developmental expression of HCMV receptors, EGFR, and integrins^34–37 determines susceptibility to infection.^33,38–40 HCMV infects spatially distinct populations of cytotrophoblasts that express α1β1 and αvβ3 integrins used as surface receptors.⁴¹How HCMV disseminates to the placenta and the early stages of pathogenesis in pregnancy are still unresolved because of the virus'' extreme host range restriction. A successful approach to overcome the obstacle to studies of HCMV in vivo has been to infect SCID mice (ie, mice with severe combined immunodeficiency) that have received xenografts of human tissues. Infection of human fetal thymus/liver under the mouse kidney capsule showed that medullary epithelial cells are prominent targets of HCMV replication.⁴² Thus, dramatic interstrain differences were evident in replication of low-passage clinical isolates and laboratory strains in thymus/liver xenografts in vivo.^42,43 The strain Toledo replicates to high titers in implants, whereas the laboratory strains AD169 and Towne, serially passaged in fibroblasts, are attenuated and fail to propagate in tissues in vivo.⁴³ AD169 lacks a 15-kb segment of viral genome that encodes at least 19 open reading frames present in the genomes of all pathogenic clinical strains.⁴⁴ A deletion mutant of Toledo lacking these sequences, although exhibiting only a minor growth defect in fibroblasts, fails to replicate in thymus/liver implants in SCID mice, evidence that genes in this region are central to infection in vivo.⁴⁵ HCMV replication in endothelial and epithelial cells correlates with determinants specified by ORFs UL128-131A,^46,47 which are highly conserved in clinical isolates⁴⁸ and which elicit neutralizing antibodies in humans.^49,50Herein, we investigated HCMV pathogenesis in infected human placental villous explants and in xenografts maintained in vivo. We used a model of human placentation to investigate the vascular effects of fetal cytotrophoblasts in human placental villi transplanted beneath the kidney capsules of SCID mice.²¹ VR1814, a clinical isolate, infected cell column cytotrophoblasts in placental explants and impaired the formation of anchoring villi in vitro. In xenografts, VR1814-infected placental cells had a severely diminished capacity to invade and form lymphatic vessels. In striking contrast, AD169 replicated poorly in villous explants. In SCID mice, AD169-infected cytotrophoblasts remodeled the resident arteries, but these were faulty. Our results show, for the first time to our knowledge, that HCMV genes dispensable for growth in culture function as determinants of pathogenesis that could contribute to vascular anomalies that originate in early placentation.