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.     

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.     

Selective Cathepsin S Inhibition Attenuates Atherosclerosis in Apolipoprotein E–Deficient Mice with Chronic Renal Disease

Chronic renal disease (CRD) accelerates the development of atherosclerosis. The potent protease cathepsin S cleaves elastin and generates bioactive elastin peptides, thus promoting vascular inflammation and calcification. We hypothesized that selective cathepsin S inhibition attenuates atherogenesis in hypercholesterolemic mice with CRD. CRD was induced by 5/6 nephrectomy in high-fat high-cholesterol fed apolipoprotein E–deficient mice. CRD mice received a diet admixed with 6.6 or 60 mg/kg of the potent and selective cathepsin S inhibitor RO5444101 or a control diet. CRD mice had significantly higher plasma levels of osteopontin, osteocalcin, and osteoprotegerin (204%, 148%, and 55%, respectively; P < 0.05), which were inhibited by RO5444101 (60%, 40%, and 36%, respectively; P < 0.05). Near-infrared fluorescence molecular imaging revealed a significant reduction in cathepsin activity in treated mice. RO5444101 decreased osteogenic activity. Histologic assessment in atherosclerotic plaque demonstrated that RO5444101 reduced immunoreactive cathepsin S (P < 0.05), elastin degradation (P = 0.01), plaque size (P = 0.01), macrophage accumulation (P < 0.01), growth differentiation factor-15 (P = 0.0001), and calcification (alkaline phosphatase activity, P < 0.01; osteocalcin, P < 0.05). Furthermore, cathepsin S inhibitor or siRNA significantly decreased expression of growth differentiation factor-15 and monocyte chemotactic protein-1 in a murine macrophage cell line and human primary macrophages. Systemic inhibition of cathepsin S attenuates the progression of atherosclerotic lesions in 5/6 nephrectomized mice, serving as a potential treatment for atherosclerosis in patients with CRD.Half of all patients with chronic renal disease (CRD) die of cardiovascular causes.^1,2 Patients with early stages of CRD who are not undergoing dialysis have cardiovascular disease (CVD) risk similar to that of patients with established coronary artery disease,³ whereas patients with end-stage renal disease, treated by dialysis, have an approximately 30 times greater CVD risk than the general population.^4,5 Despite being at elevated risk for CVD, patients with CRD have experienced limited benefits from statin treatment alone.^6,7 Hence, the need is emerging to investigate the mechanisms responsible for CVD in CRD patients to develop effective new therapies.Arterial inflammation is a key facet of atherosclerotic lesion initiation and progression.^8,9 Several molecular mechanisms participate in the response to injuries at the vascular wall and in the formation and progression of atherosclerotic lesions.¹⁰ Chronic inflammation likely accelerates atherosclerosis in patients with CRD,¹¹ and the combination of chronic inflammation and an imbalance in the calcium phosphate serum level in these patients exacerbates these processes.^12,13 In addition, CRD patients receiving hemodialysis have elevated levels of circulating proinflammatory cytokines,¹⁴ which can initiate and perpetuate the inflammation-calcification loop.Cathepsin S plays a critical role in vascular inflammation and calcification. Monocyte-derived macrophages that mediate vascular inflammation express and secrete the cysteine protease cathepsin S.¹⁵ At the basal membrane of the blood vessels, secreted cathepsin S cleaves several extracellular matrix proteins, including laminin, collagen, and, preferentially, elastin, which generate bioactive elastin peptides.^16,17 Elastin-derived peptides and fragments, also known as matrikines, can incite inflammation. Elastin peptides stimulate macrophage chemotaxis¹⁸ and promote vascular inflammation and calcification.¹⁹ In addition, cathepsin S co-localizes with regions of increased elastin breaks in atherosclerotic plaques.²⁰We previously reported that cathepsin S deficiency leads to reduced elastolytic activity and decreased vascular inflammation and calcification in the arteries of hypercholesterolemic mice with experimental CRD, providing in vivo evidence implicating cathepsin S–induced elastolysis in arterial and aortic valve calcification.²¹ To seek a clinically translatable proof of concept, the present study tested the hypothesis that treatment with a highly selective cathepsin S inhibitor attenuates inflammation and atherosclerotic lesion formation in the arteries of hypercholesterolemic mice with CRD.     

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.     

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.     

Glycosaminoglycan Regulation by VEGFA and VEGFC of the Glomerular Microvascular Endothelial Cell Glycocalyx in?Vitro

Damage to endothelial glycocalyx impairs vascular barrier function and may contribute to progression of chronic vascular disease. An early indicator is microalbuminuria resulting from glomerular filtration barrier damage. We investigated the contributions of hyaluronic acid (HA) and chondroitin sulfate (CS) to glomerular microvascular endothelial cell (GEnC) glycocalyx and examined whether these are modified by vascular endothelial growth factors A and C (VEGFA and VEGFC). HA and CS were imaged on GEnCs and their resynthesis was examined. The effect of HA and CS on transendothelial electrical resistance (TEER) and labeled albumin flux across monolayers was assessed. Effects of VEGFA and VEGFC on production and charge characteristics of glycosaminoglycan (GAG) were examined via metabolic labeling and liquid chromatography. GAG shedding was quantified using Alcian Blue. NDST2 expression was examined using real-time PCR. GEnCs expressed HA and CS in the glycocalyx. CS contributed to the barrier to both ion (TEER) and protein flux across the monolayer; HA had only a limited effect. VEGFC promoted HA synthesis and increased the charge density of synthesized GAGs. In contrast, VEGFA induced shedding of charged GAGs. CS plays a role in restriction of macromolecular flux across GEnC monolayers, and VEGFA and VEGFC differentially regulate synthesis, charge, and shedding of GAGs in GEnCs. These observations have important implications for endothelial barrier regulation in glomerular and other microvascular beds.The apical side of endothelial cells is coated with an endothelial surface layer (ESL) composed of a surface-anchored glycocalyx which is itself composed of negatively charged proteoglycans [proteins with glycosaminoglycan (GAG) side chains] and glycoproteins (glycosylated proteins) and a more loosely associated layer of adsorbed plasma proteins.¹ The endothelial glycocalyx is 200 nm to 2 μm thick (depending on the vascular bed and also on the visualization technique).²The glycocalyx mediates shear, attenuates leukocyte and platelet adhesion,² regulates systemic vascular permeability,^3–6 and allows free passage of solutes but limits passage of charged macromolecules.⁷ The ESL is damaged in reperfusion injury, inflammation and trauma, hypervolemia, atherosclerosis, and diabetes (summarized by Becker et al²). ESL thickness is reduced by hyperglycemic infusions in healthy subjects, correlating with endothelial dysfunction and an increase in vascular permeability,⁸ and in type 1 diabetes correlating with microalbuminuria,⁹ suggesting a direct link between endothelial ESL dysfunction and this dysfunction of the glomerular filtration barrier. In mice, infusions of the GAG-specific enzymes hyaluronidase, chondroitinase, and heparinase reduced the glomerular endothelial cell (GEnC) ESL thickness, resulting in reduced charge selectivity and increased macromolecular passage (proteinuria).¹⁰ In addition, proteinuria is accompanied with a loss of charge selectivity and a reduction in the core proteins decorin, fibromodulin, and versican in nonobese diabetic mice.¹¹ Salmon et al¹² demonstrated an age-related reduction in ESL in glomerular (and other) microvessels of Munich Wistar Frömter rats; this was accompanied by an increase in glomerular albumin permeability, which could be rescued by intravenous injections of lectin. Finally, using doxorubicin (Adriamycin) to induce proteinuria in mice, Jeansson et al¹³ demonstrated increased fractional clearance of larger molecules in cooled, isolated, perfused kidneys, as well as reduced charge in the glomerular filtration barrier; this was accompanied by reduced synthesis of some core proteins and GAGs in the isolated glomeruli and reduced thickness of the ESL. Taken together, these studies strongly suggest that the ESL also regulates permeability in the glomerular filtration barrier. The glomerular filtration barrier is a tightly regulated filter that restricts macromolecular protein passage while allowing filtration of water and small solutes. Dysfunction of this barrier results in proteinuria, a hallmark of kidney disease. The glomerular filtration barrier consists of a trilayer of GEnCs, a glomerular basement membrane and glomerular epithelial cells (podocytes) whose foot processes interdigitate around the glomerular microvessels. Each of these layers contributes to the regulation of glomerular macromolecular permeability.^1,14 The GEnC contribution is increasingly thought to be largely dependent on the ESL.^10,15,16We have previously studied the GEnC ESL in vitro in a human conditionally immortalized (ci) cell line and, using electron microscopy, revealed the presence of an ESL layer measuring 200 nm, which is consistent with recent sophisticated measurements of endothelial glycocalyx in vivo.¹⁷ We confirmed the presence of proteoglycan core proteins, as previously demonstrated on human GEnCs,¹⁸ and of heparan sulfate (HS), a sulfated GAG.¹⁹ Enzymatic removal of HS increased macromolecular protein passage across a monolayer by 40%, whereas electrical resistance (a measure of pathways open to water and small molecules) was not affected. Furthermore, we have shown that high-glucose conditions reduce GEnC ESL and lead to a corresponding increase in macromolecular protein passage across GEnC monolayers,²⁰ demonstrating that the GEnC glycocalyx is present in vitro and plays a functional role.Glomerular enzyme infusion studies by Jeansson et al¹⁰ implicated, in addition to removal of HS, removal of hyaluronic acid (HA) and chondroitin sulfate (CS) in increased fractional albumin clearance. HA and CS are also thought to play a role in systemic macromolecular permeability.²¹ In contrast to other GAGs, HA is not synthesized in the Golgi apparatus, but rather at the plasma membrane (by HA synthases 1 to 3), and it is not attached to core proteins (reviewed by Genasetti et al²²). Furthermore, HA is unbranched and unsulfated, and therefore it does not have a strong negative charge. CS is sulfated on assembly within the Golgi apparatus, where it becomes attached to one of its core proteins (eg, aggrecan or versican), forming a proteoglycan. In the present study, we aimed to determine the contribution of HA and CS to the GEnC glycocalyx through in vitro studies using unique human ciGEnCs and selective enzymes.Vascular endothelial growth factor A (VEGFA), originally called vascular permeability factor, is a powerful angiogenic growth factor that has profound effects on vascular endothelial behavior (as summarized by Tammela et al²³), including GEnC maintenance,²⁴ repair,²⁵ and permeability.²⁶ VEGFA is highly expressed by podocytes.²⁷ Another member of the VEGF family of proteins, VEGFC, is a lymphangiogenic growth factor that can stimulate similar pathways to those of VEGFA in both lymphatic and vascular endothelial cells.²⁸ VEGFC also is expressed by podocytes.²⁹ Podocyte–endothelial signaling through VEGF is vital for GEnC maintenance and permeability regulation. It has been postulated that the effects of VEGFA on microvessel permeability are due in part to partial degradation of the glycocalyx.³⁰ In the present study, we aimed to determine whether VEGFs can modify the GEnC glycocalyx. Our central hypothesis was that CS and HA contribute to GEnC barrier maintenance and that they are modified by VEGFs.     

Short Hairpin RNA-Mediated Knockdown of VEGFA in Müller Cells Reduces Intravitreal Neovascularization in a Rat Model of Retinopathy of Prematurity

Vascular endothelial growth factor (VEGF) A is implicated in aberrant angiogenesis and intravitreous neovascularization (IVNV) in retinopathy of prematurity (ROP). However, VEGFA also regulates retinal vascular development and functions as a retinal neural survival factor. By using a relevant ROP model, the 50/10 oxygen-induced retinopathy (OIR) model, we previously found that broad inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area compared with control IgG but also significantly reduced body weight gain in the pups, suggesting an adverse effect. Therefore, we propose that knockdown of up-regulated VEGFA in cells that overexpress it under pathological conditions would reduce IVNV without affecting physiological retinal vascular development or overall pup growth. Herein, we determined first that the VEGFA mRNA signal was located within the inner nuclear layer corresponding to CRALBP-labeled Müller cells of pups in the 50/10 OIR model. We then developed a lentiviral-delivered miR-30–embedded shRNA against VEGFA that targeted Müller cells. Reduction of VEGFA by lentivector VEGFA-shRNA–targeting Müller cells efficiently reduced 50/10 OIR up-regulated VEGFA and IVNV in the model, without adversely affecting physiological retinal vascular development or pup weight gain. Knockdown of VEGFA in rat Müller cells by lentivector VEGFA-shRNA significantly reduced VEGFR2 phosphorylation in retinal vascular endothelial cells. Our results suggest that targeted knockdown of overexpressed VEGFA in Müller cells safely reduces IVNV in a relevant ROP model.Retinopathy of prematurity (ROP) remains a leading cause of childhood blindness and is increasing in frequency in developing countries. The hypothetical proposed pathophysiological characteristics of ROP have been recently refined to be that stresses in prematurity cause delayed physiological retinal vascular development and potentially some high oxygen-induced capillary constriction that results in avascular retina.^1–4 Once supplemental oxygen is removed from the preterm infant, the retina becomes hypoxic, and hypoxia stimulates the release of angiogenic factors with growth of new blood vessels into the vitreous as intravitreous neovascularization (IVNV). Many angiogenic factors can result in pathological IVNV in animal models, such as insulin-like growth factor-1,^5,6 hepatocyte growth factor,⁷ erythropoietin,^8–10 platelet-derived growth factor,¹¹ and angiopoietins,^12,13 but vascular endothelial cell growth factor A (VEGFA) has become one of the most studied factors leading to IVNV. VEGFA mRNA was found in the retina of a preterm infant eye with severe ROP,¹⁴ and VEGFA protein was increased in vitreous from preterm infants who underwent surgery for stage 4 ROP compared with controls.¹⁵ VEGFA inhibitors reduce pathological angiogenesis in adult retinal diseases, including diabetic retinopathy^16,17 and age-related macular degeneration.^18–20 Therefore, there is reason to consider VEGFA in the pathological characteristics of human ROP. However, in the preterm infant retina, VEGFA is also important in the development of retinal blood vessels^21–23 and other organs.^24,25 After a recent clinical trial testing intravitreal delivery of a broad anti-VEGFA antibody in infants with severe ROP, there have been reports of persistent avascular retina and reactivation of IVNV with subsequent total retinal detachment, even 1 year after treatment.²⁶ In addition, by using a relevant ROP model, we found that inhibition of VEGFA bioactivity using a neutralizing antibody to rat VEGF significantly reduced IVNV area without adversely affecting physiological retinal vascular development 6 days after antibody injection, but significantly reduced body weight gain in the pups, suggesting an adverse effect.²⁷ Therefore, safer ways to inhibit pathological IVNV while preserving physiological retinal vascularization are needed.One way to target pathological IVNV is to determine the cells within the retina that overproduce VEGFA during pathological stress. In preterm infant eyes, it is not possible to safely localize where VEGFA is produced. Therefore, we used a relevant model of ROP today, the rat 50/10 oxygen-induced retinopathy (OIR) model, to localize the VEGFA signal within the retina and determine its role in pathological IVNV in ROP. This model causes features of severe ROP and produces extrauterine growth restriction, a risk for ROP in human preterm infants.²⁸ The oxygen exposure recreates arterial oxygen fluctuations similar to those experienced by infants with severe ROP.²⁹ Previously, we found that VEGFA and VEGFR2 were both increased as early as at postnatal day 8 (p8) in whole retinas from eyes of pups in the 50/10 OIR model compared with room air–raised counterparts.³⁰In the retina, several cells have been shown to produce VEGFA to support retinal development and physiological functioning. These include ganglion cells,³¹ astrocytes,³² Müller cells, and retinal pigment epithelium.³³ In pathological IVNV, the VEGFA signal has been localized to many of the same cells: Müller cells,^34,35 astrocytes,³⁶ and, possibly, ganglion cells. However, there has been disagreement as to the cell type that overproduced VEGFA to cause IVNV,^34,36 and these articles also used the mouse model of OIR that exposes pups to constant and higher oxygen levels than currently used in the management of ROP.³⁷In the current study, we generated shRNAs to VEGFA and used a lentiviral miRNA-based system to target Müller cells, where the VEGFA message was localized in the 50/10 OIR model. We tested the hypothesis that knocking down VEGFA to physiological levels in Müller cells would inhibit IVNV without adversely affecting physiological retinal vascular development.     

Development of Hepatocellular Carcinoma in a Murine Model of Nonalcoholic Steatohepatitis Induced by Use of a High-Fat/Fructose Diet and Sedentary Lifestyle

Obesity is increasingly prevalent, strongly associated with nonalcoholic liver disease, and a risk factor for numerous cancers. Here, we describe the liver-related consequences of long-term diet-induced obesity. Mice were exposed to an extended obesity model comprising a diet high in trans-fats and fructose corn syrup concurrent with a sedentary lifestyle. Livers were assessed histologically using the nonalcoholic fatty liver disease (NAFLD) activity score (Kleiner system). Mice in the American Lifestyle-Induced Obesity Syndrome (ALIOS) model developed features of early nonalcoholic steatohepatitis at 6 months (mean NAFLD activity score = 2.4) and features of more advanced nonalcoholic steatohepatitis at 12 months, including liver inflammation and bridging fibrosis (mean NAFLD activity score = 5.0). Hepatic expression of lipid metabolism and insulin signaling genes were increased in ALIOS mice compared with normal chow-fed mice. Progressive activation of the mouse hepatic stem cell niche in response to ALIOS correlated with steatosis, fibrosis, and inflammation. Hepatocellular neoplasms were observed in 6 of 10 ALIOS mice after 12 months. Tumors displayed cytological atypia, absence of biliary epithelia, loss of reticulin, alteration of normal perivenular glutamine synthetase staining (absent or diffuse), and variable α-fetoprotein expression. Notably, perivascular tumor cells expressed hepatic stem cell markers. These studies indicate an adipogenic lifestyle alone is sufficient for the development of nonalcoholic steatohepatitis, hepatic stem cell activation, and hepatocarcinogenesis in wild-type mice.Nonalcoholic fatty liver disease (NAFLD) represents one of the commonest causes of liver disease in the Western world,¹ ranging in severity from steatosis to nonalcoholic steatohepatitis (NASH) and cirrhosis.² Although simple steatosis alone is relatively benign, the presence of steatohepatitis greatly increases the risk of progression to cirrhosis, with its concomitant risk of developing hepatocellular carcinoma (HCC)³ and death.⁴ Moreover, an evolving body of literature implicates obesity with the development of cancer,⁵ including HCC.⁶ Notably, although obesity is closely associated with NAFLD, not all patients are obese, and severe NASH may develop in nonobese patients,² indicating that the interaction of factors contributing to NAFLD pathogenesis is not fully understood. A need therefore exists for murine models that accurately reflect the causative factors underpinning clinical NASH to allow for the investigation of these factors that contribute to its development and progression to cancer.Current rodent models of fatty liver disease rely on strains that carry spontaneous mutations (ob/ob⁷, db/db⁸), genetic manipulations,⁹ or formulated diets (methionine and choline deficient diet,¹⁰ high-fat diet¹¹), yet none of these models accurately reproduce the broad range of factors that contribute to the histological spectrum of human NAFLD and its sequel. More recently, combinatorial use of diets with high proportions of fat, trans-fatty acids, oxidized lipoproteins,¹² or high-fructose drinking water¹³ have resulted in patterns of liver injury closer to that observed in NASH, although aspects such as significant fibrogenesis and carcinogenesis are still lacking. Tetri et al¹⁴ added a sedentary lifestyle to a diet rich in trans-fatty acids and high-fructose corn syrup for a 16-week period and found that mice developed glucose intolerance and hepatic steatosis and inflammation. Here, we report the effects of a prolonged version (12 months) of the American Lifestyle-Induced Obesity Syndrome (ALIOS) model that more accurately represents the extended pathogenesis of NASH seen clinically.     

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.     

Junctional Adhesion Molecule A Promotes Epithelial Tight Junction Assembly to Augment Lung Barrier Function

Epithelial barrier function is maintained by tight junction proteins that control paracellular fluid flux. Among these proteins is junctional adhesion molecule A (JAM-A), an Ig fold transmembrane protein. To assess JAM-A function in the lung, we depleted JAM-A in primary alveolar epithelial cells using shRNA. In cultured cells, loss of JAM-A caused an approximately 30% decrease in transepithelial resistance, decreased expression of the tight junction scaffold protein zonula occludens 1, and disrupted junctional localization of the structural transmembrane protein claudin-18. Consistent with findings in other organs, loss of JAM-A decreased β1 integrin expression and impaired filamentous actin formation. Using a model of mild systemic endoxotemia induced by i.p. injection of lipopolysaccharide, we report that JAM-A^−/− mice showed increased susceptibility to pulmonary edema. On injury, the enhanced susceptibility of JAM-A^−/− mice to edema correlated with increased, transient disruption of claudin-18, zonula occludens 1, and zonula occludens 2 localization to lung tight junctions in situ along with a delay in up-regulation of claudin-4. In contrast, wild-type mice showed no change in lung tight junction morphologic features in response to mild systemic endotoxemia. These findings support a key role of JAM-A in promoting tight junction homeostasis and lung barrier function by coordinating interactions among claudins, the tight junction scaffold, and the cytoskeleton.To support efficient gas exchange, the lung must maintain a barrier between the atmosphere and fluid-filled tissues. Without this crucial barrier, the air spaces would flood, and gas exchange would be severely limited.^{1, 2} In acute lung injury and acute respiratory distress syndrome, fluid leakage into the lung air space is associated with increased patient mortality and morbidity.^{3, 4} Lung fluid clearance is maintained, in part, by tight junctions that regulate paracellular flux between cells.^{5, 6, 7}Tight junctions are multiprotein complexes located at sites of cell-cell contact and are composed of transmembrane, cytosolic, and cytoskeletal proteins that together produce a selective barrier to water, ions, and soluble molecules. Among the transmembrane proteins required for epithelial barrier function is the Ig superfamily protein junctional adhesion molecule A (JAM-A).^{8, 9, 10, 11} JAM-A is ubiquitously expressed and regulates several processes related to cell-cell and cell-matrix interactions, including cell migration and proliferation in addition to barrier function regulation. Specific mechanistic roles for JAM-A in regulating tight junctions continue to be elucidated.JAM-A signaling is stimulated by cis-dimerization, which provides a platform for multiple proteins to cluster in close apposition.¹² In particular, JAM-A has been shown to recruit scaffold proteins, such as zonula occludens 1 (ZO-1), ZO-2, and Par3, to tight junctions, where these proteins enhance the assembly of multiprotein junctional complexes.^{13, 14} More recently, it was demonstrated that JAM-A directly interacts with ZO-2, which then recruits other scaffold proteins, including ZO-1.¹⁵ This nucleates a core complex that includes afadin, PDZ-GEF1, and Rap2c and that stabilizes filamentous actin by repressing rhoA.¹⁵ Together, all of these activities of JAM-A promote tight junction formation and barrier function.Although JAM-A is part of the tight junction complex, the main structural determinants of the paracellular barrier are proteins known as claudins. Claudins are a family of transmembrane proteins that interact to form paracellular channels that either promote or limit paracellular ion and water flux.^{16, 17, 18} Claudins that promote flux are known collectively as pore-forming claudins, whereas claudins that limit flux are known as sealing claudins.¹⁹ In fact, there is a link between JAM-A and claudin expression because it was demonstrated that JAM-A–deficient intestinal epithelium has increased expression of two pore-forming claudins, claudin-10 and claudin-15.²⁰ Critically, increased claudin-10 and claudin-15 leads to a compromised intestinal barrier, as demonstrated by an enhanced susceptibility of JAM-A^−/− mice to dextran sulfate sodium–induced colitis.²⁰ However, it is not known whether this relationship between JAM-A and claudin expression occurs in other classes of epithelia.Several claudins are expressed by the alveolar epithelium. The most prominent alveolar claudins are claudin-3, claudin-4, and claudin-18; several additional claudins are expressed by alveolar epithelium and throughout the lung as well.^{21, 22} A central role for claudin-18 in regulating lung barrier function was demonstrated in two independently derived strains of claudin-18–deficient mice that showed altered alveolar tight junction morphologic features and increased paracellular permeability.^{23, 24} Claudin-4 also is an important part of the lung response to acute lung injury because it improves barrier function by limiting alveolar epithelial permeability and promoting lung fluid clearance.^{25, 26} Although claudin-4–deficient mice show a relatively mild baseline phenotype, these mice have impaired fluid clearance in response to ventilator-induced lung injury.²⁷ An analysis of ex vivo perfused human donor lungs revealed that increased claudin-4 was linked to increased rates of alveolar fluid clearance and decreased physiologic respiratory impairment,²⁸ further underscoring the importance of claudin regulation in promoting efficient barrier function in response to injury.Although JAM-A has a clear role in regulating gut permeability,²⁰ a recent report that wild-type and JAM-A^−/− mice show comparable levels of pulmonary edema in response to intratracheal endotoxin challenge²⁹ raises questions about potential roles for JAM-A in lung barrier function. Herein we used a combination of in vivo and in vitro approaches to assess the contributions of JAM-A to alveolar barrier function. Using a model of mild systemic endotoxemia induced by i.p. injection of Escherichia coli–derived lipopolysaccharide (LPS), we found that JAM-A^−/− mice showed greater lung edema than comparably treated wild-type mice. Greater sensitivity to injury was due to aberrant regulation of tight junction protein expression, which was recapitulated by JAM-A–depleted alveolar epithelial cells. JAM-A depletion also resulted in decreased β1 integrin protein levels and disrupted cytoskeletal assembly. Together, these effects indicated that the loss of JAM-A impaired tight junction formation, thus rendering the lung more susceptible to edema and injury.     

Mutant LRRK2 Elicits Calcium Imbalance and Depletion of Dendritic Mitochondria in Neurons

Mutations in the leucine-rich repeat kinase 2 (LRRK2) have been associated with familial and sporadic cases of Parkinson disease. Mutant LRRK2 causes in vitro and in vivo neurite shortening, mediated in part by autophagy, and a parkinsonian phenotype in transgenic mice; however, the underlying mechanisms remain unclear. Because mitochondrial content/function is essential for dendritic morphogenesis and maintenance, we investigated whether mutant LRRK2 affects mitochondrial homeostasis in neurons. Mouse cortical neurons expressing either LRRK2 G2019S or R1441C mutations exhibited autophagic degradation of mitochondria and dendrite shortening. In addition, mutant LRRK2 altered the ability of the neurons to buffer intracellular calcium levels. Either calcium chelators or inhibitors of voltage-gated L-type calcium channels prevented mitochondrial degradation and dendrite shortening. These data suggest that mutant LRRK2 causes a deficit in calcium homeostasis, leading to enhanced mitophagy and dendrite shortening.CME Accreditation Statement: This activity (“ASIP 2013 AJP CME Program in Pathogenesis”) has been planned and implemented in accordance with the Essential Areas and policies of the Accreditation Council for Continuing Medical Education (ACCME) through the joint sponsorship of the American Society for Clinical Pathology (ASCP) and the American Society for Investigative Pathology (ASIP). ASCP is accredited by the ACCME to provide continuing medical education for physicians.The ASCP designates this journal-based CME activity (“ASIP 2013 AJP CME Program in Pathogenesis”) for a maximum of 48 AMA PRA Category 1 Credit(s)™. Physicians should only claim credit commensurate with the extent of their participation in the activity.CME Disclosures: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose.Parkinson disease (PD) is a progressive neurodegenerative disease that affects both subcortical and cortical brain regions, leading to deficits in motor control and cognitive decline. Although most cases of PD are sporadic, familial mutations account for nearly 10% of patients with PD.¹ Interestingly, mutations in the leucine-rich repeat kinase 2 (LRRK2) have been identified in approximately 5% of familial PD cases and 1% of sporadic PD cases.² Although the underlying pathogenesis remains poorly understood, changes in autophagy regulation and mitochondrial homeostasis are emerging as common pathways in toxin and genetic models of PD.^3,4Previous studies have highlighted the importance of mitochondrial quality control in PD models using mitochondrial neurotoxins or proteins encoded by recessive PD-associated genes (Parkin, DJ-1, and PINK1) that traffic to the mitochondria.^5–8 Although these mitochondrial toxins and mutant proteins perturb mitochondrial respiration, degradation, and calcium buffering, less is known about how nonmitochondrially targeted PD gene products, such as LRRK2 or α-synuclein, affect mitochondrial homeostasis.^9–11 Recent studies suggest that autophagy regulation may be a point of convergence among the Parkin, PINK1, DJ-1, α-synuclein, and LRRK2 pathways.^6,12–15 Autophagy is a highly conserved, cytoplasmic recycling pathway that engulfs proteins, aggregates, and organelles in autophagosomes and traffics them to the lysosome for degradation.¹⁶ Autophagic turnover of mitochondria and aggregate-prone proteins is one way to maintain healthy, functioning neurons; however, excessive degradation may have deleterious effects leading to neurodegeneration.¹⁷ Therefore, PD-associated genes may modulate mitochondrial homeostasis by directly regulating mitochondrial function or by affecting mitochondrial degradation.⁴Although LRRK2 function remains unknown, previous studies indicate a role in regulating neurite morphological characteristics, cytoskeletal dynamics, or the endosomal-autophagic system.^12,18–25 PD-associated mutations in the GTPase domain (R1441C) and the kinase domain (G2019S) have previously been shown to cause neurite shortening and autophagy induction.^{12,19,22,23,25} LRRK2 may also play roles in synaptic transmission^26–28 and protein translation.^29,30 A recent study of skin fibroblasts from patients bearing the G2019S mutation showed altered mitochondrial function.³¹ Because LRRK2 partially associates with mitochondria^18,32 and interacts with PD-associated proteins that regulate mitochondrial stress,³³ we investigated mechanisms by which mutations in LRRK2 affect mitochondrial homeostasis in neurons.     

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.