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The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy., Introduction Over 25 million people in the United States have diabetes mellitus (1) and are prone to suffer from its devastating sequelae. The metabolic derangements seen in diabetes lead to [...]

The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1β (IL-1β), which in turn activate a well-described, [...]

Author(s): Joy M. Greer; John Puetz; Kirk R. Thomas; Mario R. Capecchi (corresponding author) Biological diversity is driven mainly by gene duplication followed by mutation and selection. This divergence in [...]

A process that directs self-induced deletion of DNA sequences as they go through the mouse male germ line has been described. The testes-specific promoter from the angiotensin-converting enzyme gene was used to drive expression of the Cre-recombinase gene. Somatic cells from the embryo-derived stem (ES) cells retained the cassette, but self-excision took place in all ES-cell-derived sperm.

Research has been conducted on apical ectodermal ridge formation. The authors have attempted to demonstrate that Wnt3, expressed throughout the limb ectoderm, is important for normal limb development and critical in establishment apical ectodermal ridge.

Mice possessing a null mutation at the Wnt3 locus fail to gastrulate and die at early stages of embryogenesis (E9.5). We have generated mice with a conditional ('fioxed') allele of Wnt3 (Wnt3c) in order to determine its roles at later stages of development. We have also generated transgenic mouse lines harboring Cre recombinase driven by the Brachyury (T) promoter as a means of specifcally removing Wnt3 in the primitive streak of embryos. We observed that mice transheterozygous for the Wnt3 null and conditional alleles (Wnt3n/c) and also possessing a 'TCre' line (Tcre14) exhibited no gastrulation anomalies but surprisingly possessed severe hindlimb outgrowth defects. We subsequently found that Tcre14 exhibited robust Cre activity in the hindlimb ectoderm. In addition, we found Wnt3 to be expressed ubiquitously in the ectoderm but not the mesenchyme of the developing limb. We demonstrate that these conditional mutants do not express markers characteristic of the apical ectodermal ridge (AER) suggesting that Wnt3 plays an important role in the establishment of this tissue. We also show that Fgf10, which is also essential for the establishment of the AER, continues to be strongly expressed in the early limb mesenchyme of these mutants suggesting that Wnt3 is likely to act downstream of this molecule. We describe further molecular characterization of the mutant hindlimbs. Finally, we describe the conditional removal of [Beta]-catenin in the limb mesenchyme vs ectoderm and argue that Wnt3 signals to the distal limb ectoderm to establish the AER.

Background: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated. Objective: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis. Methods: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP+ eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction. Results: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP+ cells per mm2 septal tissue, respectively (P <.05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice (P <.05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression. Conclusions: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis. [ABSTRACT FROM AUTHOR]

The transcription factor TBX3 plays critical roles in development and TBX3 mutations in humans cause Ulnar-mammary syndrome. Efforts to understand how altered TBX3 dosage and function disrupt the development of numerous structures have been hampered by embryonic lethality of mice bearing presumed null alleles. We generated a novel conditional null allele of Tbx3: after Cre-mediated recombination, no mRNA or protein is detectable. In contrast, a putative null allele in which exons 1-3 are deleted produces a truncated protein that is abnormally located in the cytoplasm. Heterozygotes and homozygotes for this allele have different phenotypes than their counterparts bearing a true null allele. Our observations with these alleles in mice, and the different types of TBX3 mutations observed in human ulnar-mammary syndrome, suggest that not all mutations observed in humans generate functionally null alleles. The possibility that mechanisms in addition to TBX3 haploinsufficiency may cause UMS or other malformations merits investigation in the human UMS population. [ABSTRACT FROM AUTHOR]

The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy. [ABSTRACT FROM AUTHOR]

Background: ARF nucleotide-binding site opener (ARNO) is a guanine nucleotide-exchange factor for ADP-ribosylation factor proteins. ARF nucleotide-binding site opener also binds MyD88, and small-molecule inhibition of ARNO reduces inflammation in animal models of inflammatory arthritis and acute inflammation. However, whether genetic deletion of Arno in mice reduces pathologic inflammation has not yet been reported. Furthermore, its role in the nasal cavity has yet to be investigated., Objective: To generate Arno knockout mice and to determine whether genetic loss of ARNO reduces eosinophilic inflammation in the ovalbumin (OVA) murine model of rhinitis., Methods: Arno knockout mice were generated and wild type and knockout littermates were subjected to the OVA-induced mouse model of rhinosinutitis. Eosinophilic inflammation was assessed through immunofluorescent quantification of EMBP + eosinophils in the septal mucosa and cytokine expression was assessed by quantitative polymerase chain reaction., Results: Arno knockout mice are viable and fertile without any noted deficits. Arno wild type and knockout mice subjected to the OVA-induced model of rhinitis demonstrated an average of 314.5 and 153.8 EMBP + cells per mm 2 septal tissue, respectively ( P < .05). Goblet cells per mm of basal lamina were assessed via Alcian blue and there was no statistically significant difference between Arno wild type and knockout mice. Ovalbumin-induced expression of interleukin-5 (IL-5) was significantly reduced in Arno knockout mice ( P < .05). There was no statistically significant reduction in IL-4, IL-13, or eotaxin-1 expression., Conclusions: These data demonstrate that deletion of Arno reduces eosinophilic inflammation and IL-5 expression in an OVA-induced model of rhinitis.

Rationale: Netrin-4 regulates vascular development. Identity of netrin-4 endothelial receptor and its subsequent cell functions is controversial. We previously demonstrated that the inhibition of netrin-1 canonical receptors, Unc5B and neogenin, expressed by lymphatic endothelial cells, do not suppress netrin-4-induced cell signaling and functions. Netrin family members were shown to signal through a range of receptors, including integrins (such as α3β1, α6β1, and α6β4) in nonendothelial cells., Objective: We tested whether integrins are netrin-4 receptors in the endothelium., Methods and Results: The α6β1 integrin is expressed by endothelial cells, and binds netrin-4 in a dose-dependent manner. Inhibition of α6 or β1 integrin subunits suppresses netrin-4-induced endothelial cell migration, adhesion, and focal adhesion contact. Netrin-4-stimulated phosphorylation of Src kinase family, effectors of endothelial cell migration, is also abolished by α6 or β1 inhibition. Finally, netrin-4 and α6β1 integrin expression colocalize in mouse embryonic, intestine, and tumor vasculature., Conclusions: The α6β1 integrin is a netrin-4 receptor in lymphatic endothelium and consequently represents a potential target to inhibit netrin-4-induced metastatic dissemination.