Angiopoietin-2 causes pericyte dropout in the normal retina: evidence for involvement in diabetic retinopathy

Pericyte loss is an early pathologic feature of diabetic retinopathy, consistently present in retinae of diabetic humans and animals. Because pericyte recruitment and endothelial cell survival are controlled, in part, by the angiopoietin/Tie2 ligand/receptor system, we studied the expression of angiopoietin-2 and -1 in relation to the evolution of pericyte loss in diabetic rat retinae, using quantitative retinal morphometry, and in retinae from mice with heterozygous angiopoietin deficiency (Ang-2 LacZ knock-in mice). Finally, recombinant angiopoietin-2 was injected into eyes of nondiabetic rats, and pericyte numbers were quantitated in retinal capillaries. Angiopoietin-1 protein was present in the normal maturing retina and was upregulated 2.5-fold in diabetic retinae over 3 months of diabetes. In contrast, angiopoietin-2 protein was consistently upregulated more than 30-fold in the retinae of diabetic rats, preceding the onset of pericyte loss. Heterozygous angiopoietin-2 deficiency completely prevented diabetes-induced pericyte loss and reduced the number of acellular capillary segments. Injection of angiopoietin-2 into the eyes of normal rats induced a dose-dependent pericyte loss. These data show that upregulation of angiopoietin-2 plays a critical role in the loss of pericytes in the diabetic retina.

An essential role for transmembrane TNF in the resolution of the inflammatory lesion induced by Leishmania major infection

TNF is an essential player in infections with Leishmania major, contributing to the control of the inflammatory lesion and, to a lesser degree, to parasite killing. However, the relative contribution of the soluble and transmembrane forms of TNF in these processes is unknown. To investigate the role of transmembrane TNF (mTNF) in the control of L. major infections, mTNF-knock-in (mTNF(Delta/Delta)) mice, which express functional mTNF but do not release soluble TNF, were infected with L. major, and the development of the inflammatory lesion and the immune response was compared to that occurring in L. major-infected TNF(-/-) and wild-type mice. mTNF(Delta/Delta) mice controlled the infection and resolved their inflammatory lesion as well as wild-type mice, a process associated with the early clearance of neutrophils at the site of parasite infection. In contrast, L. major-infected TNF(-/-) mice developed non-healing lesions, characterized by an elevated presence of neutrophils at the site of infection and partial control of parasite number within the lesions. Altogether, the results presented here demonstrate that mTNF, in absence of soluble TNF, is sufficient to control infection due to L. major, enabling the regulation of inflammation, and the optimal killing of Leishmania parasites at the site of infection.

PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature

The transmembrane ligand ephrinB2 and its cognate Eph receptor tyrosine kinases are important regulators of embryonic blood vascular morphogenesis. However, the molecular mechanisms required for ephrinB2 transduced cellular signaling in vivo have not been characterized. To address this question, we generated two sets of knock-in mice: ephrinB2DeltaV mice expressed ephrinB2 lacking the C-terminal PDZ interaction site, and ephrinB2(5F) mice expressed ephrinB2 in which the five conserved tyrosine residues were replaced by phenylalanine to disrupt phosphotyrosine-dependent signaling events. Our analysis revealed that the homozygous mutant mice survived the requirement of ephrinB2 in embryonic blood vascular remodeling. However, ephrinB2DeltaV/DeltaV mice exhibited major lymphatic defects, including a failure to remodel their primary lymphatic capillary plexus into a hierarchical vessel network, hyperplasia, and lack of luminal valve formation. Unexpectedly, ephrinB2(5F/5F) mice displayed only a mild lymphatic phenotype. Our studies define ephrinB2 as an essential regulator of lymphatic development and indicate that interactions with PDZ domain effectors are required to mediate its functions.

Maximal acceleration of calcium release refractoriness by β-adrenergic stimulation requires dual activation of protein kinase A and CaMKII in mouse ventricular myocytes

Time-dependent refractoriness of calcium (Ca2+) release in cardiac myocytes is an important factor in determining whether pro-arrhythmic release patterns develop. At the subcellular level of the Ca2+ spark, recent studies have suggested that recovery of spark amplitude is controlled by local sarcoplasmic reticulum (SR) refilling whereas refractoriness of spark triggering depends on both refilling and the sensitivity of the ryanodine receptor (RyR) release channels that produce sparks. Here we studied regulation of Ca2+ spark refractoriness in mouse ventricular myocytes by examining how β-adrenergic stimulation influenced sequences of Ca2+ sparks originating from individual RyR clusters. Our protocol allowed us to separately measure recovery of spark amplitude and delays between successive sparks, and data were interpreted quantitatively through simulations with a stochastic mathematical model. We found that, compared with spark sequences measured under control conditions: (1) β-adrenergic stimulation with isoproterenol accelerated spark amplitude recovery and decreased spark-to-spark delays; (2) activating protein kinase A (PKA) with forskolin accelerated amplitude recovery but did not affect spark-to-spark delays; (3) inhibiting PKA with H89 retarded amplitude recovery and increased spark- to-spark delays; (4) preventing phosphorylation of the RyR at serine 2808 with a knock-in mouse prevented the decrease in spark-to-spark delays seen with β-adrenergic stimulation; (5) inhibiting either PKA or Ca2+/calmodulin-dependent protein kinase II (CaMKII) during β-adrenergic stimulation prevented the decrease in spark-to-spark delays seen) without inhibition. The results suggest that activation of either PKA or CaMKII is sufficient to speed SR refilling, but activation of both kinases appears necessary to observe increased RyR sensitivity. The data provide novel insight into β-adrenergic regulation of Ca2+ release refractoriness in mouse myocytes.

Targeted disruption of the intracellular domain of receptor FgfrL1 in mice.

FgfrL1 is the fifth member of the fibroblast growth factor receptor (Fgfr) family. Studies with FgfrL1 deficient mice have demonstrated that the gene plays an important role during embryonic development. FgfrL1 knock-out mice die at birth as they have a malformed diaphragm and lack metanephric kidneys. Similar to the classical Fgfrs, the FgfrL1 protein contains an extracellular part composed of three Ig-like domains that interact with Fgf ligands and heparin. However, the intracellular part of FgfrL1 is not related to the classical receptors and does not possess any tyrosine kinase activity. Curiously enough, the amino acid sequence of this domain is barely conserved among different species, with the exception of three motifs, namely a dileucine peptide, a tandem tyrosine-based motif YXXΦ and a histidine-rich sequence. To investigate the function of the intracellular domain of FgfrL1, we have prepared genetically modified mice that lack the three conserved sequence motifs, but instead contain a GFP cassette (FgfrL1ΔC-GFP). To our surprise, homozygous FgfrL1ΔC-GFP knock-in mice are viable, fertile and phenotypically normal. They do not exhibit any alterations in the diaphragm or the kidney, except for a slight reduction in the number of glomeruli that does not appear to affect life expectancy. In addition, the pancreas of both FgfrL1ΔC-GFP knock-in and FgfrL1 knock-out mice do not show any disturbances in the production of insulin, in contrast to what has been suggested by recent studies. Thus, the conserved motifs of the intracellular FgfrL1 domain are dispensable for organogenesis and normal life. We conclude that the extracellular domain of the protein must conduct the vital functions of FgfrL1.

Macrophages induce neutrophil apoptosis through membrane TNF, a process amplified by Leishmania major

Neutrophils are recruited to the site of parasite inoculation within a few hours of infection with the protozoan parasite Leishmania major. In C57BL/6 mice, which are resistant to infection, neutrophils are cleared from the site of s.c. infection within 3 days, whereas they persist for at least 10 days in susceptible BALB/c mice. In the present study, we investigated the role of macrophages (MPhi) in regulating neutrophil number. Inflammatory cells were recruited by i.p. injection of either 2% starch or L. major promastigotes. Neutrophils were isolated and cultured in the presence of increasing numbers of MPhi. Extent of neutrophil apoptosis positively correlated with the number of MPhi added. This process was strictly dependent on TNF because MPhi from TNF-deficient mice failed to induce neutrophil apoptosis. Assays using MPhi derived from membrane TNF knock-in mice or cultures in Transwell chambers revealed that contact with MPhi was necessary to induce neutrophil apoptosis, a process requiring expression of membrane TNF. L. major was shown to exacerbate MPhi-induced apoptosis of neutrophils, but BALB/c MPhi were not as potent as C57BL/6 MPhi in this induction. Our results emphasize the importance of MPhi-induced neutrophil apoptosis, and membrane TNF in the early control of inflammation.

PDZ domain-binding motif regulates cardiomyocyte compartment-specific NaV1.5 channel expression and function

BACKGROUND Sodium channel NaV1.5 underlies cardiac excitability and conduction. The last 3 residues of NaV1.5 (Ser-Ile-Val) constitute a PDZ domain-binding motif that interacts with PDZ proteins such as syntrophins and SAP97 at different locations within the cardiomyocyte, thus defining distinct pools of NaV1.5 multiprotein complexes. Here, we explored the in vivo and clinical impact of this motif through characterization of mutant mice and genetic screening of patients. METHODS AND RESULTS To investigate in vivo the regulatory role of this motif, we generated knock-in mice lacking the SIV domain (ΔSIV). ΔSIV mice displayed reduced NaV1.5 expression and sodium current (INa), specifically at the lateral myocyte membrane, whereas NaV1.5 expression and INa at the intercalated disks were unaffected. Optical mapping of ΔSIV hearts revealed that ventricular conduction velocity was preferentially decreased in the transversal direction to myocardial fiber orientation, leading to increased anisotropy of ventricular conduction. Internalization of wild-type and ΔSIV channels was unchanged in HEK293 cells. However, the proteasome inhibitor MG132 rescued ΔSIV INa, suggesting that the SIV motif is important for regulation of NaV1.5 degradation. A missense mutation within the SIV motif (p.V2016M) was identified in a patient with Brugada syndrome. The mutation decreased NaV1.5 cell surface expression and INa when expressed in HEK293 cells. CONCLUSIONS Our results demonstrate the in vivo significance of the PDZ domain-binding motif in the correct expression of NaV1.5 at the lateral cardiomyocyte membrane and underline the functional role of lateral NaV1.5 in ventricular conduction. Furthermore, we reveal a clinical relevance of the SIV motif in cardiac disease.

Expression of human cathepsin L or human cathepsin V in mouse thymus mediates positive selection of T helper cells in cathepsin L knock-out mice

A genetic deficiency of the cysteine protease cathepsin L (Ctsl) in mice results in impaired positive selection of conventional CD4+ T helper cells as a result of an incomplete processing of the MHC class II associated invariant chain or incomplete proteolytic generation of positively selecting peptide ligands. The human genome encodes, in contrast to the mouse genome, for two cathepsin L proteases, namely cathepsin L (CTSL) and cathepsin V (CTSV; alternatively cathepsin L2). In the human thymic cortex, CTSV is the predominately expressed protease as compared to CTSL or other cysteine cathepsins. In order to analyze the functions of CTSL and CTSV in the positive selection of CD4+ T cells we employed Ctsl knock-out mice crossed either with transgenic mice expressing CTSL under the control of its genuine human promoter or with transgenic mice expressing CTSV under the control of the keratin 14 (K14) promoter, which drives expression to the cortical epithelium. Both human proteases are expressed in the thymus of the transgenic mice, and independent expression of both CTSL and CTSV rescues the reduced frequency of CD4+ T cells in Ctsl-deficient mice. Moreover, the expression of the human cathepsins does not change the number of CD4+CD25+Foxp3+ regulatory T cells, but the normalization of the frequency of conventional CD4+ T cell in the transgenic mice results in a rebalancing of conventional T cells and regulatory T cells. We conclude that the functional differences of CTSL and CTSV in vivo are not mainly determined by their inherent biochemical properties, but rather by their tissue specific expression pattern.

Increased numbers of spontaneous SCLC metastasis in absence of NK cells after subcutaneous inoculation of different SCLC cell lines into pfp/rag2 double knock out mice

Spontaneous metastases in small cell lung cancer (SCLC) occur regularly in patients but seldom if any in conventional xenograft mouse models. To overcome this problem, SCLC cells were grafted subcutaneously onto pore forming protein and recombination activating gene 2 double knock out (pfp/rag2) mice and in severe combined immunodeficient (scid) mice. Primary tumours grew well in both mouse strains, while metastases occurred frequently in the pfp/rag2 mice and infrequently in scid mice. Hence NK cells, which are inactive in pfp/rag2 mice, play an important role in SCLC metastasis formation in xenograft models. This observation is in agreement with clinical studies, where a high NK cell number in the blood is correlated with a better prognosis of the patient.

Early development of human lymphomas in a prostate cancer xenograft program using triple knock-out immunocompromised mice

BACKGROUND There is an urgent need for preclinical models of prostate cancer; however, clinically relevant patient-derived prostate cancer xenografts (PDXs) are demanding to establish. METHODS Sixty-seven patients who were undergoing palliative transurethral surgery or radical prostatectomy for histologically confirmed, clinically relevant prostate cancer were included in the study. Fresh prostate cancer tissue was identified by frozen analysis in 48 patients. The cancer tissue was transplanted subcutaneously and under the renal capsule of NSG and NOG mice supplemented with human testosterone. All growing PDXs were evaluated by histology and immunohistochemistry. RESULTS Early assessment of the animals at least three months after transplantation included 27/48 (56.3%) eligible PDX cohorts. PDX growth was detected in 10/27 (37%) mouse cohorts. Eight of the ten PDXs were identified as human donor derived lymphomas, including seven Epstein Barr virus (EBV)-positive diffuse large B-cell lymphomas and one EBV-negative peripheral T-cell lymphoma. One sample consisted of benign prostatic tissue, and one sample comprised a benign epithelial cyst. Prostate cancer was not detected in any of the samples. CONCLUSIONS Tumors that arise within the first three months after prostate cancer xenografting may represent patient-derived EBV-positive lymphomas in up to 80% of the early growing PDXs when using triple knockout NSG immunocompromised mice. Therefore, lymphoma should be excluded in prostate cancer xenografts that do not resemble typical prostatic adenocarcinoma. Prostate 9999: XX-XX, 2014. © 2015 Wiley Periodicals, Inc.

Genomic knock-out of rancRNAs in the archaeon H. volcanii

As translation is the final step in gene expression it is particularly important to understand the processes involved in translation regulation. It was shown in the last years that a class of RNA, the non-protein-coding RNAs (ncRNAs), is involved in regulation of gene expression via various mechanisms [1]. Herein included is the prominent example of gene silencing caused by micro RNAs (miRNAs) and small interfering RNAs (siRNAs). Almost all of these ncRNA discovered so far target the mRNA in order to modulate protein biosynthesis, this is rather unexpected considering the crucial role of the ribosome during gene expression. However, recent data from our laboratory showed that there is a new class of RNAs among the well-studied ncRNAs that target the ribosome itself [2,3]. These so called ribosome-associated ncRNAs (rancRNAs) have an impact on translation regulation, mainly by interfering / modulating the rate of protein biosynthesis. Recent studies show the presence of small regulatory RNAs (sRNAs) in archaea which are involved in many biological processes including stress response and metabolic regulation [4]. To date the biological function and the targets of these archaeal sRNAs are only described for a few examples. There are reports of sRNAs binding to the 5’ as well as to the 3’ of mRNAs [5,6]. In addition to these findings, a tRNA derived fragment (tRF) of Valine tRNA was found in a genomic screen of RNAs associated with the ribosome in H. volcanii in our laboratory [3]. This Valine tRF seems to be processed in a stress-dependent manner and showed in vitro binding to the ribosome and inhibited in vitro translation. These results showed that Valine tRF is capable to regulate translation in H. volcanii by targeting the ribosome. The main goal of this project is to identify and describe novel potential regulatory rancRNAs in H. volcanii with the focus on intergenic candidates. Northern blot analyses already revealed interactions with the ribosome and showed differential expression patterns in response to stress conditions. To investigate the biological relevance of some of the ribosome-associated ncRNA candidates, knock-out and phenotypic characterization studies are done. The genomic knock out of a hypothetical ORF (198nt), where one putative rancRNA candidate (46nt) named IG33 was detected in the library at the beginning of the ORF, showed interesting growth phenotype under specific stress conditions. Furthermore a strain with an introduced start to stop codon mutation in this hypothetical ORF still shows the same phenotype indicating that rather the missing protein than the missing sRNA causes this growth phenotype.

Knock-out of Arabidopsis metal transporter gene IRT1 results in iron deficiency accompanied by cell differentiation defects

IRT1 and IRT2 are members of the Arabidopsis ZIP metal transporter family that are specifically induced by iron deprivation in roots and act as heterologous suppressors of yeast mutations inhibiting iron and zinc uptake. Although IRT1 and IRT2 are thought to perform redundant functions as root-specific metal transporters, insertional inactivation of the IRT1 gene alone results in typical symptoms of iron deficiency causing severe leaf chlorosis and lethality in soil. The irt1 mutation is characterized by specific developmental defects, including a drastic reduction of chloroplast thylakoid stacking into grana and lack of palisade parenchyma differentiation in leaves, reduced number of vascular bundles in stems, and irregular patterns of enlarged endodermal and cortex cells in roots. Pulse labeling with 59Fe through the root system shows that the irt1 mutation reduces iron accumulation in the shoots. Short-term labeling with 65Zn reveals no alteration in spatial distribution of zinc, but indicates a lower level of zinc accumulation. In comparison to wild-type, the irt1 mutant responds to iron and zinc deprivation by altered expression of certain zinc and iron transporter genes, which results in the activation of ZIP1 in shoots, reduction of ZIP2 transcript levels in roots, and enhanced expression of IRT2 in roots. These data support the conclusion that IRT1 is an essential metal transporter required for proper development and regulation of iron and zinc homeostasis in Arabidopsis.

The cytidine deaminases AID and APOBEC-1 exhibit distinct functional properties in a novel yeast selectable system

Activation-induced cytidine deaminase (AID) is indispensable for immunoglobulin maturation by somatic hypermutations and class switch recombination and is supposed to deaminate cytidines in DNA, while its homolog APOBEC-1 edits apolipoprotein (apo) B mRNA by cytidine deamination. We studied the editing activity of APOBEC-1 and AID in yeast using the selectable marker Gal4 linked to its specific inhibitor protein Gal80 via an apo B cassette (Gal4-C) or via the variable region of a mouse immunoglobulin heavy chain gene (Gal4-VH). Expression of APOBEC-1 induced C to U editing in up to 15% of the Gal4-C transcripts, while AID was inactive in this reaction even in the presence of the APOBEC-1 complementation factor. After expression of APOBEC-1 as well as AID approximately 10(-3) of yeast cells survived low stringency selection and expressed beta-galactosidase. Neither AID nor APOBEC-1 mutated the VH sequence of Gal4-VH, and consequently the yeast colonies did not escape high stringent selection. AID, however, induced frequent plasmid recombinations that were only rarely observed with APOBEC-1. In conclusion, AID cannot substitute APOBEC-1 to edit the apo B mRNA, and the expression of AID in yeast is not sufficient for the generation of point mutations in a highly transcribed Gal4-VH sequence. Cofactors for AID induced somatic hypermutations of immunoglobulin variable regions, that are present in B cells and a variety of non-B cells, appear to be missing in yeast. In contrast to APOBEC-1, AID alone does not exhibit an intrinsic specificity for its target sequences.