A neutralizable dimeric anti-thrombin aptamer with potent anticoagulant activity in mice.

Heparin-induced thrombocytopenia (HIT) is a complication caused by administration of the anticoagulant heparin. Although the number of patients with HIT has drastically increased because of coronavirus disease 2019 (COVID-19), the currently used thrombin inhibitors for HIT therapy do not have antidotes to arrest the severe bleeding that occurs as a side effect; therefore, establishment of safer treatments for HIT patients is imperative. Here, we devised a potent thrombin inhibitor based on bivalent aptamers with a higher safety profile via combination with the antidote. Using an anti-thrombin DNA aptamer M08s-1 as a promising anticoagulant, its homodimer and heterodimer with TBA29 linked by a conformationally flexible linker or a rigid duplex linker were designed. The dimerized M08s-1-based aptamers had about 100-fold increased binding affinity to human and mouse thrombin compared with the monomer counterparts. Administration of these bivalent aptamers into mice revealed that the anticoagulant activity of the dimers significantly surpassed that of an approved drug for HIT treatment, argatroban. Moreover, adding protamine sulfate as an antidote against the most potent bivalent aptamer completely suppressed the anticoagulant activity of the dimer. Emerging potent and neutralizable anticoagulant aptamers will be promising candidates for HIT treatment with a higher safety profile.

Intestinal Diffuse Large B-Cell Lymphoma in a Patient with Systemic Lupus Erythematosus.

A 44-year-old Japanese woman with systemic lupus erythematosus (SLE) presented to our hospital with abdominal pain. Radiological and endoscopic examinations led to the diagnosis of diffuse large B-cell lymphoma of the jejunum, which was subsequently resected. Patients with SLE reportedly have an increased risk of non-Hodgkin lymphoma, as demonstrated by our patient. Hence, lymphoma should be considered in the differential diagnosis of neoplastic lesions emerging in SLE patients. In addition, flow cytometry using endoscopically biopsied fragments is useful for the immediate diagnosis of lymphoma, leading to timely and accurate preoperative staging.

WNT7A/B promote choroidal neovascularization.

Perturbations in WNT signaling are associated with congenital eye disorders, including familial exudative vitreoretinopathy and Norrie disease. More recently, activation of the WNT pathway has also been shown to be associated with age-related macular degeneration (AMD). In this study, we identified that in choroidal neovascular membranes from AMD patients, ß-catenin is activated specifically in the vascular endothelium, suggesting that WNT promotes pathologic angiogenesis by directly affecting vascular endothelial cells. WNT7B has been shown to be important during eye development for regression of the fetal hyaloid vasculature. However, it has not yet been established whether WNT7A and/or WNT7B are involved in neovascular AMD pathogenesis. Here, we show that WNT7A and WNT7B increase the proliferation of human dermal microvascular endothelial cells in a dose-dependent manner. Both WNT7A and WNT7B also stimulated vascular sprouting from mouse choroidal explants in vitro. To evaluate in vivo relevance, we generated mice systemically deficient in Wnt7a and/or Wnt7b. Genetic deletion of both Wnt7a and Wnt7b decreased the severity of laser injury-induced choroidal neovascularization (CNV), while individual deletion of either Wnt7a or Wnt7b did not have a significant effect on CNV, suggesting that WNT7A and WNT7B have redundant pro-angiogenic roles in vivo. Cumulatively, these findings identify specific WNT isoforms that may play a pathologic role in CNV as observed in patients with neovascular AMD. Although the source of increased WNT7A and/or WNT7B in CNV requires further investigation, WNT signaling may be a potential target for therapeutic intervention if these results are demonstrated to be relevant in human disease.

NAMPT-Mediated NAD(+) Biosynthesis Is Essential for Vision In Mice.

Photoreceptor death is the endpoint of many blinding diseases. Identifying unifying pathogenic mechanisms in these diseases may offer global approaches for facilitating photoreceptor survival. We found that rod or cone photoreceptor-specific deletion of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the major NAD(+) biosynthetic pathway beginning with nicotinamide, caused retinal degeneration. In both cases, we could rescue vision with nicotinamide mononucleotide (NMN). Significantly, retinal NAD(+) deficiency was an early feature of multiple mouse models of retinal dysfunction, including light-induced degeneration, streptozotocin-induced diabetic retinopathy, and age-associated dysfunction. Mechanistically, NAD(+) deficiency caused metabolic dysfunction and consequent photoreceptor death. We further demonstrate that the NAD(+)-dependent mitochondrial deacylases SIRT3 and SIRT5 play important roles in retinal homeostasis and that NAD(+) deficiency causes SIRT3 dysfunction. These findings demonstrate that NAD(+) biosynthesis is essential for vision, provide a foundation for future work to further clarify the mechanisms involved, and identify a unifying therapeutic target for diverse blinding diseases.

Impaired autophagy in macrophages promotes inflammatory eye disease.

Autophagy is critical for maintaining cellular homeostasis. Organs such as the eye and brain are immunologically privileged. Here, we demonstrate that autophagy is essential for maintaining ocular immune privilege. Deletion of multiple autophagy genes in macrophages leads to an inflammation-mediated eye disease called uveitis that can cause blindness. Loss of autophagy activates inflammasome-mediated IL1B secretion that increases disease severity. Inhibition of caspase activity by gene deletion or pharmacological means completely reverses the disease phenotype. Of interest, experimental uveitis was also increased in a model of Crohn disease, a systemic autoimmune disease in which patients often develop uveitis, offering a potential mechanistic link between macrophage autophagy and systemic disease. These findings directly implicate the homeostatic process of autophagy in blinding eye disease and identify novel pathways for therapeutic intervention in uveitis.

Injury-Mediated Vascular Regeneration Requires Endothelial ER71/ETV2.

OBJECTIVE: Comprehensive understanding of the mechanisms regulating angiogenesis might provide new strategies for angiogenic therapies for treating diverse physiological and pathological ischemic conditions. The E-twenty six (ETS) factor Ets variant 2 (ETV2; aka Ets-related protein 71) is essential for the formation of hematopoietic and vascular systems. Despite its indispensable function in vessel development, ETV2 role in adult angiogenesis has not yet been addressed. We have therefore investigated the role of ETV2 in vascular regeneration. APPROACH AND RESULTS: We used endothelial Etv2 conditional knockout mice and ischemic injury models to assess the role of ETV2 in vascular regeneration. Although Etv2 expression was not detectable under steady-state conditions, its expression was readily observed in endothelial cells after injury. Mice lacking endothelial Etv2 displayed impaired neovascularization in response to eye injury, wounding, or hindlimb ischemic injury. Lentiviral Etv2 expression in ischemic hindlimbs led to improved recovery of blood perfusion with enhanced vessel formation. After injury, fetal liver kinase 1 (Flk1), aka VEGFR2, expression and neovascularization were significantly upregulated by Etv2, whereas Flk1 expression and vascular endothelial growth factor response were significantly blunted in Etv2-deficient endothelial cells. Conversely, enforced Etv2 expression enhanced vascular endothelial growth factor-mediated endothelial sprouting from embryoid bodies. Lentiviral Flk1 expression rescued angiogenesis defects in endothelial Etv2 conditional knockout mice after hindlimb ischemic injury. Furthermore, Etv2(+/-); Flk1(+/-) double heterozygous mice displayed a more severe hindlimb ischemic injury response compared with Etv2(+/-) or Flk1(+/-) heterozygous mice, revealing an epistatic interaction between ETV2 and FLK1 in vascular regeneration. CONCLUSIONS: Our study demonstrates a novel obligatory role for the ETV2 in postnatal vascular repair and regeneration.

IL10-driven STAT3 signalling in senescent macrophages promotes pathological eye angiogenesis.

Macrophage dysfunction plays a pivotal role during neovascular proliferation in diseases of ageing including cancers, atherosclerosis and blinding eye disease. In the eye, choroidal neovascularization (CNV) causes blindness in patients with age-related macular degeneration (AMD). Here we report that increased IL10, not IL4 or IL13, in senescent eyes activates STAT3 signalling that induces the alternative activation of macrophages and vascular proliferation. Targeted inhibition of both IL10 receptor-mediated signalling and STAT3 activation in macrophages reverses the ageing phenotype. In addition, adoptive transfer of STAT3-deficient macrophages into eyes of old mice significantly reduces the amount of CNV. Systemic and CD163(+) eye macrophages obtained from AMD patients also demonstrate STAT3 activation. Our studies demonstrate that impaired SOCS3 feedback leads to permissive IL10/STAT3 signalling that promotes alternative macrophage activation and pathological neovascularization. These findings have significant implications for our understanding of the pathobiology of age-associated diseases and may guide targeted immunotherapy.

Activation of Wnt/ß-catenin signaling in Muller glia protects photoreceptors in a mouse model of inherited retinal degeneration.

The canonical Wnt/ß-catenin ("Wnt") pathway is an essential signaling cascade in the embryonic central nervous system (CNS) that regulates neuronal differentiation and survival. Loss of Wnt signaling in developing and adult tissue has been implicated in numerous CNS diseases, but the precise role of Wnt in regulating neuronal survival, and how its absence could lead to disease, is not understood. In this study, we investigated the effect of Wnt activation on neuronal survival in the adult retina, and identified cellular and molecular mediators. Pan-retinal Wnt signaling activation using Wnt3a induced functional and morphological rescue of photoreceptor neurons in the rd10 mouse model of retinal degeneration. Furthermore, Wnt activation using constitutively active ß-catenin specifically targeted to Muller glia increased photoreceptor survival and reduced markers of glial and neuronal remodeling. Wnt-induced photoreceptor protection was associated with elevated levels of the prosurvival protein Stat3, and was reduced by shRNA-mediated knock-down of Stat3, indicating cross-talk between survival pathways. Therefore, these data increase our understanding of the role of Wnt signaling in the retina, and identify radial Muller glia as important cellular mediators of Wnt activity.

Endothelial cell FGF signaling is required for injury response but not for vascular homeostasis.

Endothelial cells (ECs) express fibroblast growth factor receptors (FGFRs) and are exquisitely sensitive to FGF signals. However, whether the EC or another vascular cell type requires FGF signaling during development, homeostasis, and response to injury is not known. Here, we show that Flk1-Cre or Tie2-Cre mediated deletion of FGFR1 and FGFR2 (Fgfr1/2(Flk1-Cre) or Fgfr1/2(Tie2-Cre) mice), which results in deletion in endothelial and hematopoietic cells, is compatible with normal embryonic development. As adults, Fgfr1/2(Flk1-Cre) mice maintain normal blood pressure and vascular reactivity and integrity under homeostatic conditions. However, neovascularization after skin or eye injury was significantly impaired in both Fgfr1/2(Flk1-Cre) and Fgfr1/2(Tie2-Cre) mice, independent of either hematopoietic cell loss of FGFR1/2 or vascular endothelial growth factor receptor 2 (Vegfr2) haploinsufficiency. Also, impaired neovascularization was associated with delayed cutaneous wound healing. These findings reveal a key requirement for cell-autonomous EC FGFR signaling in injury-induced angiogenesis, but not for vascular homeostasis, identifying the EC FGFR signaling pathway as a target for diseases associated with aberrant vascular proliferation, such as age-related macular degeneration, and for modulating wound healing without the potential toxicity associated with direct manipulation of systemic FGF or VEGF activity.

Impaired cholesterol efflux in senescent macrophages promotes age-related macular degeneration.

Pathologic angiogenesis mediated by abnormally polarized macrophages plays a central role in common age-associated diseases such as atherosclerosis, cancer, and macular degeneration. Here we demonstrate that abnormal polarization in older macrophages is caused by programmatic changes that lead to reduced expression of ATP binding cassette transporter ABCA1. Downregulation of ABCA1 by microRNA-33 impairs the ability of macrophages to effectively efflux intracellular cholesterol, which in turn leads to higher levels of free cholesterol within senescent macrophages. Elevated intracellular lipid polarizes older macrophages to an abnormal, alternatively activated phenotype that promotes pathologic vascular proliferation. Mice deficient for Abca1, but not Abcg1, demonstrate an accelerated aging phenotype, whereas restoration of cholesterol efflux using LXR agonists or miR-33 inhibitors reverses it. Monocytes from older humans with age-related macular degeneration showed similar changes. These findings provide an avenue for therapeutic modulation of macrophage function in common age-related diseases.

The Wnt/ß-catenin pathway cross-talks with STAT3 signaling to regulate survival of retinal pigment epithelium cells.

Wnt/ß-catenin signaling is an essential pathway that regulates numerous cellular processes, including cell survival. The molecular mechanisms contributing to pro-survival Wnt signaling are mostly unknown. Signal transducer and activator of transcription proteins (STATs) are a well-described family of transcription factors. STAT3 induces expression of anti-apoptotic genes in many tissues and is a downstream mediator of protective growth factors and cytokines. In this study, we investigated whether pro-survival Wnt signaling is mediated by STAT3. The Wnt3a ligand activated Wnt signaling in the retinal pigment epithelium ARPE-19 cell line and significantly increased the viability of cells exposed to oxidative stress. Furthermore, Wnt3a increased STAT3 activation and nuclear translocation, as measured by an antibody against phosphorylated STAT3. Reducing STAT3 levels with siRNA eliminated Wnt3a-dependent protection from oxidative stress. Together, these data demonstrate a previously unknown link between Wnt3a-mediated activation of STAT3 and cell survival, and indicate cross-talk between two important pro-survival signaling pathways.

The Wnt signaling pathway protects retinal ganglion cell 5 (RGC-5) cells from elevated pressure.

The Wnt pathway is an essential signaling cascade that regulates survival and differentiation in the retina. We recently demonstrated that retinal ganglion cells (RGCs) have constitutively active Wnt signaling in vivo. However, the role of Wnt in RGC viability or function is unknown. In this study, we investigated whether Wnt protects the retinal ganglion cell line RGC-5 from elevated pressure, oxidative stress, and hypoxia injuries. Expression of RGC marker genes in the RGC-5 cultures was confirmed by immunocytochemistry and PCR. We demonstrated that the Wnt3a ligand significantly reduced pressure-induced caspase activity in RGC-5 cells (n = 5, P = 0.03) and decreased the number of TUNEL-positive cells (n = 5, P = 0.0014). Notably, Wnt3a-dependent protection was reversed by the Wnt signaling inhibitor Dkk1. In contrast, Wnt3a did not protect RGC-5 cells from oxidative stress or hypoxia. Furthermore, Wnt3a significantly increased growth factor expression in the presence of elevated pressure but not in the presence of oxidative stress and hypoxia. These results indicate that Wnt3a induces injury-specific survival pathways in RGC-5 cells, potentially by upregulating neuroprotective growth factors. Therefore, activation of the Wnt pathway by Wnt3a could be investigated further as a tool to develop novel molecular therapeutic strategies for the prevention of RGC death in retinal disease.

Analysis of Dickkopf3 interactions with Wnt signaling receptors.

Wnt signaling regulates essential biological processes ranging from embryogenesis to neurodegeneration. Recently, we demonstrated that Dickkopf3 (Dkk3) is a pro-survival glycoprotein that positively modulates Wnt signaling. An important step in understanding the mechanism of action of Dkk3 is identifying its interacting proteins in the Wnt pathway. In this study, we used a series of biochemical and functional assays to investigate the interaction between Dkk3 and the Wnt pathway receptors Kremen1 (Krm1), Kremen 2 (Krm2) and low-density lipoprotein receptor-related protein 6 (LRP6). Here, we report that, contrary to previous studies, Dkk3 interacts with Krm1 and Krm2. However, Dkk3 did not interact with, or alter expression of, LRP6. Blocking protein glycosylation did not alter the interaction between Dkk3 and Krm proteins. Additionally, Krm2 abolished Dkk3-mediated potentiation of Wnt signaling. Therefore, our data establish that Krm proteins are novel binding partners of Dkk3 and suggest a mechanism by which Dkk3 potentiates Wnt signaling.

Characterization of Wnt signaling during photoreceptor degeneration.

PURPOSE: The Wnt pathway is an essential signaling cascade that regulates multiple processes in developing and adult tissues, including differentiation, cellular survival, and stem cell proliferation. The authors recently demonstrated altered expression of Wnt pathway genes during photoreceptor death in rd1 mice, suggesting an involvement for Wnt signaling in the disease process. In this study, the authors investigated the role of Wnt signaling in retinal degeneration. METHODS: The Wnt signaling reporter mouse line Tcf-LacZ was crossed with retinal degeneration rd1 mice, and beta-galactosidase expression was used to localize Wnt signaling during photoreceptor death. To analyze the role of Wnt signaling activation, primary mixed retinal cultures were prepared, and XTT and TUNEL assays were used to quantify cell death. Luciferase reporter assays were used to measure Wnt signaling. RESULTS: The canonical Wnt signaling pathway was activated in Müller glia and the ganglion cell layer during rod photoreceptor degeneration in rd1/Tcf-LacZ mice. Wnt signaling was confirmed in cultured primary Müller glia. Furthermore, Wnt signaling activators protected photoreceptors in primary retinal cultures from H(2)O(2)-induced oxidative stress. The Wnt ligands Wnt5a, Wnt5b, Wnt10a, and Wnt13 were expressed in the degenerating retina and are candidate Wnt signaling activators in vivo. CONCLUSIONS: This study is the first demonstration that Wnt signaling is activated in the degenerating retina and that it protects retinal cultures from oxidative stress. These data suggest that Wnt signaling is a component of the glial protective response during photoreceptor injury. Therefore, inducing Wnt activation, alone or in combination with growth factors, may increase the threshold for apoptosis and halt or delay further photoreceptor degeneration.

Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina.

BACKGROUND: The Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk) family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1) and Dickkopf 2 (Dkk2) are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3) protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death. RESULTS: Dkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis. CONCLUSION: These data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina.

Beneficial effect of laserpitin, a coumarin compound from Angelica keiskei, on lipid metabolism in stroke-prone spontaneously hypertensive rats.

Recently, we found that 4-hydroxyderricin, one of the major chalcones in Angelica keiskei extract (an ethyl acetate extract from the yellow liquid of stems), suppressed increases in systolic blood pressure and reduced both serum very low-density lipoprotein levels and liver triglyceride content in stroke-prone spontaneously hypertensive rats (SHRSP). In the present study, we have isolated laserpitin, a characteristic coumarin, from the A. keiskei extract and examined the effect of dietary laserpitin on blood pressure and lipid metabolism in SHRSP. Six-week-old male SHRSP were fed diets containing 0.1% laserpitin for 7 weeks with free access to the diet and water. Bodyweight gain was reduced by dietary laserpitin after 4 weeks through to 7 weeks without any significant change in daily food intake. Serum total cholesterol, phospholipid and apolipoprotein (apo) E levels were significantly increased, which was due to significant increases in cholesterol, phospholipid and apoE contents in the low- and high-density lipoprotein (LDL and HDL, respectively) fractions. These results suggest that dietary laserpitin increases serum apoE-HDL levels. In the liver, significant decreases in relative liver weight and triglyceride content were found after treatment with laserpitin for 7 weeks. An investigation of hepatic mRNA expression of proteins involved in lipid metabolism indicated that a significant decrease in hepatic triglyceride lipase may be responsible for the increase in serum HDL levels and also indicated that a marked decrease in adipocyte determination and differentiation factor 1 may be responsible, at least in part, for the decrease in hepatic triglyceride content. In conclusion, dietary laserpitin produces increases in serum HDL levels, especially apoE-HDL, and decreases in the hepatic triglyceride content in SHRSP.