Minocycline inhibits alkali burn-induced corneal neovascularization in mice

The purpose of this study was to investigate the effects of minocycline on alkali burn-induced corneal neovascularization (CNV). A total of 105 mice treated with alkali burns were randomly divided into three groups to receive intraperitoneal injections of either phosphate buffered saline (PBS) or minocycline twice a day (60 mg/kg or 30 mg/kg) for 14 consecutive days. The area of CNV and corneal epithelial defects was measured on day 4, 7, 10, and14 after alkali burns. On day 14, a histopathological examination was performed to assess morphological change and the infiltration of polymorphonuclear neutrophils (PMNs). The mRNA expression levels of vascular endothelial growth factor (VEGF) and its receptors (VEGFRs), basic fibroblast growth factor (bFGF), matrix metalloproteinases (MMPs), interleukin-1α, 1β, 6 (IL-1α, IL-1β, IL-6) were analyzed using real-time quantitative polymerase chain reaction. The expression of MMP-2 and MMP-9 proteins was determined by gelatin zymography. In addition, enzyme-linked immunosorbent assay was used to analyze the protein levels of VEGFR1, VEGFR2, IL-1β and IL-6. Minocycline at a dose of 60 mg/kg or 30 mg/kg significantly enhanced the recovery of the corneal epithelial defects more than PBS did. There were significant decreases of corneal neovascularization in the group of high-dosage minocycline compared with the control group at all checkpoints. On day 14, the infiltrated PMNs was reduced, and the mRNA expression of VEGFR1, VEGFR2, bFGF, IL-1β, IL-6, MMP-2, MMP-9, -13 as well as the protein expression of VEGFR2, MMP-2, -9, IL-1β, IL-6 in the corneas were down-regulated with the use of 60 mg/kg minocycline twice a day. Our results showed that the intraperitoneal injection of minocycline (60 mg/kg b.i.d.) can significantly inhibit alkali burn-induced corneal neovascularization in mice, possibly by accelerating corneal wound healing and by reducing the production of angiogenic factors, inflammatory cytokines and MMPs.     

Genetic modification of iron metabolism in mice affects the gut microbiota

The composition of the gut microbiota is affected by environmental factors as well as host genetics. Iron is one of the important elements essential for bacterial growth, thus we hypothesized that changes in host iron homeostasis, may affect the luminal iron content of the gut and thereby the composition of intestinal bacteria. The iron regulatory protein 2 (Irp2) and one of the genes mutated in hereditary hemochromatosis Hfe , are both proteins involved in the regulation of systemic iron homeostasis. To test our hypothesis, fecal metal content and a selected spectrum of the fecal microbiota were analyzed from Hfe-/-, Irp2-/- and their wild type control mice. Elevated levels of iron as well as other minerals in feces of Irp2-/- mice compared to wild type and Hfe-/- mice were observed. Interestingly significant variation in the general fecal-bacterial population-patterns was observed between Irp2-/- and Hfe-/- mice. Furthermore the relative abundance of five species, mainly lactic acid bacteria, was significantly different among the mouse lines. Lactobacillus (L.) murinus and L. intestinalis were highly abundant in Irp2-/- mice, Enterococcus faecium species cluster and a species most similar to Olsenella were highly abundant in Hfe-/- mice and L. johnsonii was highly abundant in the wild type mice. These results suggest that deletion of iron metabolism genes in the mouse host affects the composition of its intestinal bacteria. Further studying the relationship between gut microbiota and genetic mutations affecting systemic iron metabolism in human should lead to clinical implications.     

Human tears reveal insights into corneal neovascularization

Corneal neovascularization results from the encroachment of blood vessels from the surrounding conjunctiva onto the normally avascular cornea. The aim of this study is to identify factors in human tears that are involved in development and/or maintenance of corneal neovascularization in humans. This could allow development of diagnostic tools for monitoring corneal neovascularization and combination monoclonal antibody therapies for its treatment. In an observational case-control study we enrolled a total of 12 patients with corneal neovascularization and 10 healthy volunteers. Basal tears along with reflex tears from the inferior fornix, superior fornix and using a corneal bath were collected along with blood serum samples. From all patients, ocular surface photographs were taken. Concentrations of the pro-angiogenic cytokines interleukin (IL)-6, IL-8, Vascular Endothelial Growth Factor (VEGF), Monocyte Chemoattractant Protein 1 (MCP-1) and Fas Ligand (FasL) were determined in blood and tear samples using a flow cytometric multiplex assay. Our results show that the concentration of pro-angiogenic cytokines in human tears are significantly higher compared to their concentrations in serum, with highest levels found in basal tears. Interestingly, we could detect a significantly higher concentration of IL- 6, IL-8 and VEGF in localized corneal tears of patients with neovascularized corneas when compared to the control group. This is the first study of its kind demonstrating a significant difference of defined factors in tears from patients with neovascularized corneas as compared to healthy controls. These results provide the basis for future research using animal models to further substantiate the role of these cytokines in the establishment and maintenance of corneal neovascularization.     

Role of senescent fibroblasts on alkali-induced corneal neovascularization

Cellular senescence acts as a potent regulator of tumor suppression and fibrosis limitation; however, its contribution and crosstalk with neovascularization during normal wound healing has not been examined. Here, we explored the role of senescent fibroblasts on neovascularization with a mouse model of alkali-induced corneal wound healing. Senescent cells accumulated in corneal stroma from day 7 to 27 after alkali burn and peaked on day 14, which was consistent with the development of corneal neovascularization (CNV). In vitro and in vivo assays confirmed that the senescent cells were derived primarily from activated corneal fibroblasts. Furthermore, senescent corneal fibroblasts exhibited enhanced synthesis and secretion of extracellular matrix-degrading enzymes (matrix metalloproteinases 2, 3, and 14 and tissue- and urokinase-type plasminogen activators) and angiogenic factors (vascular endothelial growth factor) and decreased expression of anti-angiogenic factors (pigment epithelium-derived factor and thrombospondins), which supported the proliferation, migration, and promotion of tube formation of vascular endothelial cells. Intrastromal injection of premature senescent fibroblasts induced CNV earlier than that of normal fibroblasts, while matrix metalloproteinase inhibitors blocked the early onset of senescent cell-induced CNV. Therefore, senescent fibroblasts promoted the alkali-induced CNV partially via the enhanced secretion of matrix metalloproteases.     

Blockade of TREM-1 prevents vitreoretinal neovascularization in mice with oxygen-induced retinopathy

In pathological retinal neovascularization (RNV) disorders, the retina is infiltrated by activated leukocytes and macrophages. Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, activates monocytes and macrophages and plays an important role in cancer, autoimmune and other inflammation-associated disorders. Hypoxia-inducible TREM-1 is involved in cancer angiogenesis but its role in RNV remains unclear. Here, to close this gap, we evaluated the role of TREM-1 in RNV using a mouse model of oxygen-induced retinopathy (OIR). We found that hypoxia induced overexpression of TREM-1 in the OIR retinas compared to that of the room air group. TREM-1 was observed specifically in areas of pathological RNV, largely colocalizing with macrophage colony-stimulating factor (M-CSF) and CD45- and Iba-1-positive cells. TREM-1 blockade using systemically administered first-in-class ligand-independent TREM-1 inhibitory peptides rationally designed using the signaling chain homooligomerization (SCHOOL) strategy significantly (up to 95%) reduced vitreoretinal neovascularization. The peptides were well-tolerated when formulated into lipopeptide complexes for peptide half-life extension and targeted delivery. TREM-1 inhibition substantially downregulated retinal protein levels of TREM-1 and M-CSF suggesting that TREM-1-dependent suppression of pathological angiogenesis involves M-CSF. Targeting TREM-1 using TREM-1-specific SCHOOL peptide inhibitors represents a novel strategy to treat retinal diseases that are accompanied by neovascularization including retinopathy of prematurity.     

Attenuation of retinal vascular development and neovascularization in PECAM-1-deficient mice

Platelet-endothelial cell adhesion molecule-1 (PECAM-1/CD31) is expressed on the surface of endothelial cells (EC) at high levels with important roles in angiogenesis and inflammation. However, the physiological role PECAM-1 plays during vascular development and angiogenesis remains largely unknown. Here we determined the role of PECAM-1 in the postnatal development of retinal vasculature and retinal neovascularization during oxygen-induced ischemic retinopathy (OIR) using PECAM-1-deficient (PECAM-1−/−) mice. A significant decrease in retinal vascular density was observed in PECAM-1−/− mice compared with PECAM-1+/+ mice. This was attributed to a decreased number of EC in the retinas of PECAM-1−/− mice. An increase in the rate of apoptosis was observed in retinal vessels of PECAM-1−/− mice, which was compensated, in part, by an increase in the rate of proliferation. However, the development and regression of hyaloid vasculature were not affected in the absence of PECAM-1. We did not observe a significant defect in astrocytes, the number of endothelial tip cell filopodias, and the rate of developing retinal vasculature progression in PECAM-1−/− mice. However, we observed aberrant organization of arterioles and venules, decreased secondary branching, and dilated vessels in retinal vasculature of PECAM-1−/− mice. In addition, retinal neovascularization was attenuated in PECAM-1−/− mice during OIR despite an expression of VEGF similar to that of PECAM-1+/+ mice. Mechanistically, these changes were associated with an increase in EphB4 and ephrin B2, and a decrease in eNOS, expression in retinal vasculature of PECAM-1−/− mice. These results suggest that PECAM-1 expression and its potential interactions with EphB4/ephrin B2 and eNOS are important for survival, migration, and functional organization of EC during retinal vascular development and angiogenesis.     

Genetic modification in floriculture

Micro-propagation, embryo rescue, mutagenesis via chemical or irradiation means and in vitro inter-specific hybridisation methods have been used by breeders in the floriculture industry for many years. In the past 20 years these enabling technologies have been supplemented by genetic modification methods. Though many genes of potential utility to the floricultural industry have been identified, and much has been learnt of the genetic factors and molecular mechanisms underlying phenotypes of great importance to the industry, there are only flower colour modified varieties of carnation and rose in the marketplace. To a large extent this is due to unique financial barriers to market entry for genetically modified varieties of flower crops, including use of technology fees and costs of regulatory approval.     

Topical dimethyl sulfoxide inhibits corneal neovascularization and stimulates corneal repair in rabbits following acid burn

Neovascularization of the cornea is characterized by the growth of blood vessels caused by imbalances between angiogenic and anti-angiogenic factors. We investigated whether the expression of Vascular endothelial growth factor (VEGF), Vascular endothelial growth factor receptor (VEGF), Vascular endothelial growth inhibitor (VEGI) receptors, as well as topical drug treatments, participate in regulating corneal neovascularization after corneal damage and remodeling. We used 72 mature male New Zealand rabbits. Corneal burns were induced by hydrofluoric acid under general anesthesia. The rabbits then were treated with indomethacin or dimethyl sulfoxide (DMSO). The animals were euthanized on days 2, 7 and 14 after injury. Each cornea was fixed with 10% neutral formalin. On days 2, 7 and 14, VEGF, flk1/KDR and flt1/fms were strongly expressed in the epithelial, stromal and inflammatory cells, but not in the corneal endothelial cells. On day 7, newly formed blood vessels were observed growing toward the center of the cornea. In the control, indomethacin treated, DMSO–treated, and indomethacin + DMSO–treated animals, VEGI, VEGF, and the receptors, flk1/KDR, flt1/fms and flt4, were expressed at different densities in the neovascular regions. This was particularly evident in the indomethacin- and indomethacin + DMSO–treated groups on days 7 and 14, compared to day 2. Treatment with VEGF and DMSO stimulated repair of corneal damage. We suggest that VEGI in the endothelial cells of neovascularized cornea may act as a signaling protein that promotes balance between cell proliferation and apoptosis. Topical administration of DMSO inhibited corneal neovascularization more effectively than indomethacin.     

Functional analysis of Asb-1 using genetic modification in mice

The Asbs are a family of ankyrin repeat proteins that, along with four other protein families, contain a C-terminal SOCS box motif, which was first identified in the suppressor of cytokine signaling (SOCS) proteins. While it is clear that the SOCS proteins are involved in the negative regulation of cytokine signaling, the biological roles of the other SOCS box-containing families are unknown. We have investigated Asb-1 function by generating mice that lack this protein, as well as mice that overexpress full-length or truncated Asb-1 in a wide range of tissues. Although Asb-1 is expressed in multiple organs, including the hematopoietic compartment in wild-type mice, Asb-1(-/-) mice develop normally and exhibit no anomalies of mature blood cells or their progenitors. While most organs in these mice appear normal, the testes of Asb-1(-/-) mice display a diminution of spermatogenesis with less complete filling of seminiferous tubules. In contrast, the widespread overexpression of Asb-1 in the mouse has no apparent deleterious effects.     

Inhibition of VEGF expression and corneal neovascularization by siRNA targeting cytochrome P450 4B1

Injury to the cornea leads to formation of mediators that initiate and amplify inflammatory responses and neovascularization. Among these are lipid mediators generated by a cytochrome P450 (CYP) enzyme identified as CYP4B1. Increased corneal CYP4B1 expression increases limbal angiogenic activity through the production of 12-hydroxyeicosatrienoic acid (12-HETrE), a potent inflammatory and angiogenic eicosanoid. We used siRNA duplexes targeting CYP4B1 to substantiate the link between CYP4B1 expression, 12-HETrE production and angiogenesis in a model of suture-induced corneal neovascularization. Intrastromal sutures induced a time-dependent neovascular response which was significantly attenuated by CYP4B1-specific siRNAs but not by nonspecific siRNA. CYP4B1 mRNA was reduced by 60% and 12-HETrE's levels were barely detected in corneal homogenates from eyes treated with the CYP4B1-specific siRNA. The decreased neovascular response in CYP4B1 siRNA-treated eyes was associated with a 75% reduction in corneal VEGF mRNA levels. Transfection of rabbit corneal epithelial cells with CYP4B1 cDNA induced VEGF expression. Conversely, treatment with CYP4B1 siRNA or addition of a CYP4B1 inhibitor significantly decreased VEGF mRNA levels; addition of 12-HETrE potently increased them. The results strongly implicate the corneal CYP4B1 as a component of the inflammatory and neovascular cascade initiated by injury and further suggest that CYP4B1-12-HETrE is a proximal regulator of VEGF expression.     

Macrophage inflammatory protein-2 and vascular endothelial growth factor regulate corneal neovascularization induced by infection with Pseudomonas aeruginosa in mice

Pseudomonas aeruginosa ocular infection causes extensive corneal neovascularization. The purpose of the present study was to investigate the role of the angiogenic factors macrophage inflammatory protein-2 (MIP-2) and vascular endothelial growth factor (VEGF) in the regulation of corneal neovascularization during P. aeruginosa ocular infection. After administering anti-MIP-2 antibody or control antibody, mouse corneas were challenged with P. aeruginosa. The expression of MIP-2 and VEGF was detected using an ELISA from ocular homogenates. Corneal neovascularization was examined by histology. The cellular sources of MIP-2 and VEGF were identified by immunohistochemistry. In addition, protein expression of MIP-2 and VEGF in isolated corneas was measured to determine the ability of the cornea to produce these two mediators. Results showed that the expression of MIP-2 and VEGF was significantly (P < 0.05) elevated after bacterial infection, and high levels of these two mediators paralleled the extensive corneal neovascularization seen at later stages of the infection. Anti-MIP-2 antibody treatment resulted in a significant (P < 0.05) reduction in VEGF expression and in corneal neovascularization. Both corneal resident cells and infiltrating neutrophils had the ability to produce MIP-2 and VEGF after stimulation. The present study demonstrates that both MIP-2 and VEGF are important mediators in the regulation of corneal neovascularization caused by P. aeruginosa infection, and that MIP-2 regulates the production of VEGF.     

Genetic modification of prenatal lethality and dilated cardiomyopathy in Mn superoxide dismutase mutant mice.

Mn superoxide dismutase (MnSOD), a mitochondrial antioxidant enzyme, has been shown to be essential for animal survival. MnSOD mutant mice (Sod2-/- mice) on the CD1 background develop severe dilated cardiomyopathy and usually die within 10 d after birth. To characterize better the phenotype and understand the mechanism of superoxide-mediated tissue damage in Sod2-/- mice, congenic Sod2-/- mice on inbred backgrounds were generated to ensure genetic homogeneity. When generated on a C57BL/6J background (B6), more than half of the fetuses develop severe dilated cardiomyopathy by embryonic day 15 and die in the uterus. Those that survive to term usually die within 24 h. In contrast, Sod2-/- mice on DBA/2J (D2) and B6D2F1 (B6D2F1) backgrounds develop normally throughout gestation and do not develop dilated cardiomyopathy. However, the D2 mice do develop a severe metabolic acidosis and survive for only up to 12 d after birth. B6D2F1) mice have a milder form of metabolic acidosis and can survive for up to 3 weeks. The marked difference in lifespans and the development of dilated cardiomyopathy in the B6 but not the D2 or B6D2F1 backgrounds indicate the possible existence of genetic modifiers that provide protection to the developing hearts in the absence of MnSOD.     

Chemical modification of corn fiber with ion-exchanging groups

Corn fiber was chemically modified with ion-exchanging groups to prepare water-soluble polysaccharides. The soluble fractions were dialyzed using dialysis tubing (1 kDa) and the material retained inside the tubing was filtered through 10 kDa membranes to separate into fractions with molar mass of 1–10 kDa and greater than 10 kDa. The yield of solubilized material of molar mass higher than 10 kDa (47%) and 1–10 kDa (17%) obtained by sulfonation in the presence of NaOH under vacuum was greater than the yields of the treatment at the ambient pressure (43% and 2%) and also in experiments run with only KOH (40% and 5%) or NaOH (38% and 5%) at ambient pressure. The sugar analysis indicated that they were typical glucuronogalactoarabinoxylans containing 46–57% d-xylose (Xyl), 25–33% l-arabinose (Ara) and 6–12% d-galactose (Gal).     

Genetic analysis of photosynthesis and productivity in corn

Summary The genetic relationships among different inbred stocks of corn with respect to different indices of photosynthetic efficiency and plant productivity was assessed by means of diallel graphs. The salient feature of the current study was apparent overdominance for some important indices of photosynthetic efficiency, viz. photosynthetic rate/unit leaf area (at the silking stage), total chlorophyll content and chlorophyll a content, as well as for total dry matter production and economic yield. The results could be explained in terms of complementary gene action and the multiplicative effects of the sub-components that comprise these traits. However, complete dominance was recorded for photosynthetic rate (at the grain filling period), leaf area/plant, number of leaves/plant, number of leaves above the ear, ratio of chlorophyll a/b and harvest index. In the majority of cases graphic analysis was possible only when a number of epistatic parents were omitted from the analysis. Although the position of the array points for different indices of photosynthetic efficiency was consistent across the years, the ontogenetic and seasonal differences in the genetic behaviour of parents, meaning thereby, different loci being active at different stages and seasons, was apparent. The same was true for the epistatic parents. Non-allelic interaction of the genes rather than the non-random distribution of the genes among the parents seemed to be the most common cause of disturbance in the W_r/V_r relationship.Inbred stock MG 115 was identified to be the most promising because it embodied an efficient photosynthetic machinery by virtue of increasing the number of alleles for a majority of the indices of photosynthetic efficiency, thereby enabling it to register the highest biological productivity and economic yield. Further, inbred stocks MG 138, MG 121, and MG 125 were also promising for different photosynthetic parameters.     

Genetic analysis of Pycr1 and Pycr2 in mice

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.     

Genetic analysis of low lambda 1 chain production in mice

Several commonly used strains of laboratory mice from Charles River Laboratories were found to produce extremely low or undetectable levels of serum immunoglobulins bearing lambda 1 light chain (lambda 1 Ig). Individual CF-1, CD-1, and CFW random-bred mice were tested for serum lambda 1 levels, lambda 1-specific anti-NP responses, and genomic polymorphisms at the lambda 1 locus. In all cases, a complete correlation among these parameters was observed. The results indicated that nearly all CFW, greater than 70% of CD-1 but none of the CF-1 mice produced low levels of lambda 1 light chain. The low lambda 1 Ig production is due to a genetic defect either similar or identical to that observed in SJL mice. The data suggest that the lambda 1 locus of CD-1, CFW, and SJL mice are derived from a common ancestor. We also surveyed lambda 1 Ig production in a series of wild mice. Mice producing low lambda 1 Ig were frequently observed. The wild mice with low lambda 1 Ig levels were captured in diverse geographic areas, including Europe, Middle East, Asia, and South America. Preliminary study suggests that the defect in the wild mice is different from that of SJL, CD-1, or CFW mice and implies that other mechanisms regulate lambda 1 Ig production in wild mice.     

Spontaneous corneal opacities in laboratory mice

Corneal opacities were observed in numerous strains and stocks of laboratory mice (Mus musculus) from different microbiological environments. The opacities were characterized as acute and chronic inflammatory lesions of the corneal epithelium and anterior corneal stroma, including corneal ulcers and erosions, acute keratitis, stromal neovascularization and mineralization of the basement membrane zone. Some strains and stocks of mice from barrier-reared colonies had a high incidence of corneal opacities [DBA/2 (29.1%), C3H (16.2%), CF1 (16.2%) and BALB/c (10.0%)] while others had a lower incidence [CD-1 (4.3%) and C57BL/6 (4.1%)]. Axenic and gnotobiotic mice had a very low incidence of corneal opacities (1.6%). An experimental study demonstrated that twice weekly cage cleaning would reduce the incidence of corneal opacities to a very low level. A bacterial product, such as ammonia, is proposed as a significant factor in the pathogenesis of spontaneous corneal opacities in laboratory mice.     

Genetic modification of Zymomonas mobilis

The bacterium Zymomonas mobilis is a potentially useful organism for the commercial production of ethanol as it is capable of more than double the rate of alcohol production by yeast. However, industrial application of this bacterium has been restricted in part due to the disadvantages of its limited substrate range (glucose, fructose and sucrose) and by-product formation. Progress in strain improvement and genetic manipulation of this ethanologen is reviewed. Methodologies for gaining reproducible gene transfer in Z. mobilis have recently been developed. Genetic modification has led to its growth on the additional substrates lactose and mannitol. Additionally, a range of by-product negative mutants have also been isolated. Further interest has focused on transfer of Z. mobilis genes to other fermentive organisms in order to gain enhanced product formation. Overall, these genetic approaches should lead to development of novel strains of Z. mobilis and other genera, capable of the use of starch, cellulose and xylan in a manner attractive for industrial ethanol production, besides facilitating over production of products from E. coli strains with enhanced capability to grow at high density.