BEL1-LIKE HOMEODOMAIN4 regulates chlorophyll accumulation, chloroplast development, and cell wall metabolism in tomato fruit.

Tomato (Solanum lycopersicum) is a model plant for studying fruit development and ripening. In this study, we found that down-regulation of a tomato bell-like homeodomain 4 (SlBL4) resulted in a slightly darker-green fruit phenotype and increased accumulation of starch, fructose, and glucose. Analysis of chlorophyll content and TEM observations was consistent with these phenotypes, indicating that SlBL4 was involved in chlorophyll accumulation and chloroplast formation. Ripened fruit of SlBL4-RNAi plants had noticeably decreased firmness, larger intercellular spaces, and thinner cell walls than the wild-type. RNA-seq identified differentially expressed genes involved in chlorophyll metabolism, chloroplast development, cell wall metabolism, and carotenoid metabolism. ChIP-seq identified (G/A) GCCCA (A/T/C) and (C/A/T) (C/A/T) AAAAA (G/A/T) (G/A) motifs. SlBL4 directly inhibited the expression of protoporphyrinogen oxidase (SlPPO), magnesium chelatase H subunit (SlCHLD), pectinesterase (SlPE), protochlorophyllide reductase (SlPOR), chlorophyll a/b binding protein 3B (SlCAB-3B), and homeobox protein knotted 2 (TKN2). In contrast, it positively regulated the expression of squamosa promoter binding protein-like colorless non-ripening (LeSPL-CNR). Our results indicate that SlBL4 is involved in chlorophyll accumulation, chloroplast development, cell wall metabolism, and the accumulation of carotenoids during tomato fruit ripening, and provide new insights for the transcriptional regulation mechanism of BELL-mediated fruit growth and ripening.

Inhibition of integrin α5ß1 ameliorates VEGF-induced retinal neovascularization and leakage by suppressing NLRP3 inflammasome signaling in a mouse model.

PURPOSE: To assess the effect of inhibiting integrin α5ß1 by ATN-161 on vascular endothelial growth factor (VEGF)-induced neovascularization (NV) and leakage causing retinal detachment in adult Tet/opsin/VEGF transgenic mice, and characterize the underlying mechanism of its function. METHOD: Retinas from adult Tet/opsin/VEGF transgenic mice and human retinal endothelial cells (HRECs) exposed to VEGF (treated with ATN-161 or PBS) were used to carry out immunofluorescence, RT-PCR and western blot to examine expression levels of integrin α5ß1 and the NACHT, LRR, and PYD domains-containing protein 3 (NLRP3) inflammasome. Retinal frozen section analysis was used to assess NV and leakage causing retinal detachment. RESULTS: In comparison to normal-treated mice, doxycycline-treated Tet/opsin/VEGF transgenic mice showed severe retinal detachment and higher integrin α5ß1 expression. Furthermore, the retinal detachment was inhibited significantly by ATN-161. Additionally, ATN-161 treatment was associated with a conspicuous reduction in NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved caspase-1, and mature interleukin-1ß expression levels in the retinas of Tet/opsin/VEGF transgenic mice treated with doxycycline as well as in HRECs exposed to VEGF. CONCLUSION: ATN-161, an antagonist of integrin α5ß1, is a promising treatment for retinal neovascularization (RNV), and its retinal protection role appears to take effect through inhibition of NLRP3 inflammasome activity.

ATN-161 as an Integrin α5ß1 Antagonist Depresses Ocular Neovascularization by Promoting New Vascular Endothelial Cell Apoptosis.

BACKGROUND ATN-161 (Ac-PHSCN-NH2), an antagonist of integrin α5ß1, has shown an important influence in inhibiting tumor angiogenesis and metastasis of other tumor types. However, the mechanism of action of ATN-161 and whether it can inhibit ocular neovascularization (NV) are unclear. This study investigated the role of ATN-161 in regulating ocular angiogenesis in mouse models and explored the underlying signaling pathway. MATERIAL AND METHODS An oxygen-induced retinopathy (OIR) mouse model and a laser-induced choroidal neovascularization (CNV) mouse model were used to test integrin a5b1 expression and the effect of ATN-161 on ocular NV by immunofluorescence staining, Western blot analysis, and flat-mount analysis. The activation of nuclear factor-κB (NF-κB), matrix metalloproteinase-2/9 (MMP-2/9), and cell apoptosis were detected by immunofluorescence staining, Western blot, real-time RT-PCR, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). The cell proliferation was detected by BrdU labeling. RESULTS In OIR and CNV mice, the protein expression level of integrin α5ß1 increased compared with that in age-matched controls. The mice given ATN-161 had significantly reduced retinal neovascularization (RNV) and CNV. Blocking integrin a5b1 by ATN-161 strongly inhibited nuclear factor-κB (NF-κB) activation and matrix metalloproteinase-2/9 (MMP-2/9) expression and promoted cell apoptosis, but the effect of ATN-161 on proliferation in CNV mice was indirect and required the inhibition of neovascularization. Inhibiting NF-κB activation by ammonium pyrrolidinedithiocarbamate (PDTC) reduced RNV and promoted cell apoptosis in ocular NV. CONCLUSIONS Blocking integrin α5ß1 by ATN-161 reduced ocular NV by inhibiting MMP-2/MMP-9 expression and promoting the cell apoptosis of ocular NV.

Interleukin-17A neutralization alleviated ocular neovascularization by promoting M2 and mitigating M1 macrophage polarization.

Neovascularization (NV), as a cardinal complication of several ocular diseases, has been intensively studied, and research has shown its close association with inflammation and immune cells. In the present study, the role of interleukin-17A (IL-17A) in angiogenesis in the process of ocular NV both in vivo and in vitro was investigated. Also, a paracrine role of IL-17A was demonstrated in the crosstalk between endothelial cells and macrophages in angiogenesis. In the retinas of mice with retinopathy of prematurity, the IL-17A expression increased significantly at postnatal day 15 (P15) and P18 during retinal NV. Mice given IL-17A neutralizing antibody (NAb) developed significantly reduced choroidal NV and retinal NV. Studies on vascular endothelial growth factor (VEGF) over-expressing mice suggested that IL-17A modulated NV through the VEGF pathway. Furthermore, IL-17A deficiency shifted macrophage polarization toward an M2 phenotype during retinal NV with significantly reduced M1 cytokine expression compared with wild-type controls. In vitro assays revealed that IL-17A treated macrophage supernatant gave rise to elevated human umbilical vascular endothelial cell proliferation, tube formation and VEGF receptor 1 and receptor 2 expression. Therefore, IL-17A could potentially serve as a novel target for treating ocular NV diseases. The limitation of this study involved the potential mechanisms, such as which transcription accounted for macrophage polarization and how the subsequent cytokines were modulated when macrophages were polarized. Further studies need to be undertaken to definitively determine the extent to which IL-17A neutralizing anti-angiogenic activity depends on macrophage modulation compared with anti-VEGF treatment.

Neutralization of IL-23 depresses experimental ocular neovascularization.

Interleukin-23 (IL-23) is a heterodimeric cytokine that consists of p19, a novel subunit, and p40, which is shared by IL-12. IL-23 has been demonstrated to play an important role in autoimmunity and tumor growth. However, the role of IL-23 in ocular neovascularization (NV) diseases remains unclear. In this study, we explored the role of IL-23 in the processing of retinal and choroidal neovascularization (RNV and CNV). We found a significantly higher expression of IL-23 in the retinas with oxygen-induced retinopathy (OIR), and after neutralizing IL-23, the mRNA and protein levels of the angiogenic factors vascular endothelial growth factor receptor (VEGFR)1/FLT-1, VEGFR2/FLK-1, placental growth factor (PIGF), endothelial-specific receptor tyrosine kinase (Tie2), inducible nitric-oxide synthase (iNOS), matrix metalloproteinase (MMP) 2 and MMP9 were significantly down regulated, while the opposite trend was found for the anti-angiogenic molecules chemokine (C-X-C motif) ligand (CXCL) 9 and CXCL10. IL-23 blockade caused less NV in both the RNV and CNV mouse models. In addition, our in vitro assay showed that IL-23 alone is able to increase the ability of endothelial cells to form tubes. Our findings suggest that targeting IL-23 could be a potential therapy for RNV and CNV diseases.

Class I TCP in fruit development: much more than growth.

Fruit development can be viewed as the succession of three main steps consisting of the fruit initiation, growth and ripening. These processes are orchestrated by different factors, notably the successful fertilization of flowers, the environmental conditions and the hormones whose action is coordinated by a large variety of transcription factors. Among the different transcription factor families, TEOSINTE BRANCHED 1, CYCLOIDEA, PROLIFERATING CELL FACTOR (TCP) family has received little attention in the frame of fruit biology despite its large effects on several developmental processes and its action as modulator of different hormonal pathways. In this respect, the comprehension of TCP functions in fruit development remains an incomplete puzzle that needs to be assembled. Building on the abundance of genomic and transcriptomic data, this review aims at collecting available TCP expression data to allow their integration in the light of the different functional genetic studies reported so far. This reveals that several Class I TCP genes, already known for their involvement in the cell proliferation and growth, display significant expression levels in developing fruit, although clear evidence supporting their functional significance in this process remains scarce. The extensive expression data compiled in our study provide convincing elements that shed light on the specific involvement of Class I TCP genes in fruit ripening, once these reproductive organs acquire their mature size. They also emphasize their putative role in the control of specific biological processes such as fruit metabolism and hormonal dialogue.

SlZHD17 is involved in the control of chlorophyll and carotenoid metabolism in tomato fruit.

Chlorophylls and carotenoids are essential and beneficial substances for both plant and human health. Identifying the regulatory network of these pigments is necessary for improving fruit quality. In a previous study, we identified an R2R3-MYB transcription factor, SlMYB72, that plays an important role in chlorophyll and carotenoid metabolism in tomato fruit. Here, we demonstrated that the SlMYB72-interacting protein SlZHD17, which belongs to the zinc-finger homeodomain transcription factor family, also functions in chlorophyll and carotenoid metabolism. Silencing SlZHD17 in tomato improved multiple beneficial agronomic traits, including dwarfism, accelerated flowering, and earlier fruit harvest. More importantly, downregulating SlZHD17 in fruits resulted in larger chloroplasts and a higher chlorophyll content. Dual-luciferase, yeast one-hybrid and electrophoretic mobility shift assays clarified that SlZHD17 regulates the chlorophyll biosynthesis gene SlPOR-B and chloroplast developmental regulator SlTKN2 in a direct manner. Chlorophyll degradation and plastid transformation were also retarded after suppression of SlZHD17 in fruits, which was caused by the inhibition of SlSGR1, a crucial factor in chlorophyll degradation. On the other hand, the expression of the carotenoid biosynthesis genes SlPSY1 and SlZISO was also suppressed and directly regulated by SlZHD17, which induced uneven pigmentation and decreased the lycopene content in fruits with SlZHD17 suppression at the ripe stage. Furthermore, the protein-protein interactions between SlZHD17 and other pigment regulators, including SlARF4, SlBEL11, and SlTAGL1, were also presented. This study provides new insight into the complex pigment regulatory network and provides new options for breeding strategies aiming to improve fruit quality.

Overexpression of the KNOX gene Tkn4 affects pollen development and confers sensitivity to gibberellin and auxin in tomato.

The knotted1-like homeobox genes not only regulate the formation and differentiation of meristems and vascular system but are also involved in biosynthesis and signal transduction of diverse plant hormones in tomato. Here, we showed that a knotted1-like homeobox gene Tkn4 is required for pollen and pollen tube growth when this gene is overexpressed in tomato. Pollen grains in the Tkn4 overexpressed plants (Tkn4-OX) germinated quicker than those in the wild-type (WT) plant cultured in vitro in germination media. The percentage of fruit set was higher in Tkn4-OX than in WT plants and the transgenic plants showed an ordered inflorescence. Tkn4-OX seedlings also exhibited sensitivity to gibberellins (GA) and auxins. RNA sequencing results showed that the expression of genes related to sugar, cell wall-modification, microtubule-associated vesicular transport for pollen growth, GA and auxin synthesis were significantly changed. Hence, Tkn4 contributes to a function in the development of pollen and pollen tube and the regulation of phytohormones to participate in plant growth. These results provided a potential application value for agricultural improvement to enhance the rate of fruit set in tomato.

Flt3 Regulation in the Mononuclear Phagocyte System Promotes Ocular Neovascularization.

Fms-like tyrosine kinase 3 (Flt3), a tyrosine kinase receptor expressed in CD34+ hematopoietic stem/progenitor cells, is important for both normal myeloid and lymphoid differentiation. It has been implicated in mice and humans for potential multilineage differentiation. We found that mice deficient in Flt3 or mice that received an Flt3 inhibitor (AC220) showed significantly reduced areas of ischemia-induced retinal neovascularization (RNV) and laser-induced choroidal NV (CNV) (P < 0.05). Increased Flt3 expression at the protein level was detected in retinas of oxygen-induced retinopathy (OIR) mice at P15 and P18 during retinal NV (RNV) progression. We subsequently found that macrophages (Mphi) polarization was regulated at the site of CNV in Flt3-deficient mice. Flow cytometry analysis demonstrated that Flt3 deficiency shifted Mphi polarization towards an M2 phenotype during RNV with significant reduction in M1 cytokine expression when compared to the wild-type controls (P < 0.05). Based on the above findings, we concluded that Flt3 inhibition alleviated ocular NV by promoting a Mphi polarization shift towards the M2 phenotype. Therapies targeting Flt3 may provide a new approach for the treatment of ocular NV.

Endocan Blockade Suppresses Experimental Ocular Neovascularization in Mice.

Purpose: Ocular neovascularization (NV) is a pathologic process characterized by the proliferation and infiltration of various types of cells such as RPE, glial, and endothelial cells, which interact with proangiogenic factors and inflammatory cytokines. Endocan is known to be enriched in retinal endothelial tip cells under hypoxia, but the effect of endocan on ocular NV progression is largely unknown. In this study, we investigated the role of endocan in the ocular NV pathologic process and the possible mechanisms involved. Methods: In the eyes of mice with oxygen-induced retinopathy (OIR); choroidal NV (CNV); and rhodopsin promoter (rho)/VEGF transgenic mice, endocan expression was assessed by quantitative real-time PCR (RT-PCR) and Western blot. In vivo, a specific functional antibody was used to neutralize endocan and ocular NV levels were evaluated by RT-PCR, Western blot and immunostaining of flat-mounts. In vitro, the effect of endocan on human retinal microvascular endothelial cell (HREC) tube formation was observed using a routine method. Results: Endocan was significantly elevated in these three experimental mice models. Endocan blockade with the neutralizer intravitreal injection not only suppressed the area of retinal, choroidal and subretinal NV, but also resulted in a decrease in several angiogenesis-associated molecules. Recombinant endocan protein (rhEndocan) was found to induce tube formation on HRECs directly. Conclusions: The current data suggest that endocan is a potential therapeutic or an additional target for retinal and subretinal NV diseases.

Identification of different macrophage subpopulations with distinct activities in a mouse model of oxygen-induced retinopathy.

The aim of the present study was to characterize the phenotypic shift, quantity and role changes in different subgroups of retinal macrophages in a mouse model of oxygen-induced retinopathy (OIR). The mRNA expression levels of macrophage M1 and M2 subgroup marker genes and polarization-associated genes were analyzed by RT-qPCR. The number of M1 and M2 macrophages in our mouse model of OIR was analyzed by flow cytometry at different time points during the progression of OIR. Immunofluorescence whole mount staining of the retinas of mice with OIR was performed at different time points to examine the influx of macrophages, as well as the morphological characteristics and roles of M1 and M2 macrophages. An increased number of macrophages was recruited during the progression of angiogenesis in the retinas of mice with OIR due to the pro-inflammatory microenvironment containing high levels of cell adhesion and leukocyte transendothelial migration molecules. RT-qPCR and flow cytometric analysis at different time points revealed a decline in the number of M1 cells from a significantly high level at post-natal day (P)13 to a relatively normal level at P21, as well as an increase in the number of M2 cells from P13 to P21 in the mice with OIR, implicating a shift of macrophage polarization towards the M2 subtype. Immunofluorescence staining suggested that the M1 cells interacted with endothelial tip cells at the vascular front, while M2 cells embraced the emerging vessels and bridged the neighboring vessel sprouts. Thus, our data indicate that macrophages play an active role in OIR by contributing to the different steps of neovascularization. Our findings indicate that tissue macrophages may be considered as a potential target for the anti-angiogenic therapy of ocular neovascularization disease.

Pigment epithelium-derived factor regulates glutamine synthetase and l-glutamate/l-aspartate transporter in retinas with oxygen-induced retinopathy.

PURPOSE: A predominant function of Müller cells is to regulate glutamate levels, but these cells are compromised in oxygen-induced retinopathy. The aim of this study was to investigate the role of pigment epithelium-derived factor (PEDF) in regulating glutamate levels in retina under hypoxia. MATERIALS AND METHODS: One-week-old C57BL/6J mice were exposed to 75% oxygen for 5 days and then kept in room air for another 5 days to establish the oxygen-induced retinopathy (OIR) mouse model. Mice received intravitreous injections of 2 µg PEDF or vehicle on postnatal (P)12 and P14, respectively. Antibody against interleukin-1Beta (IL-1ß) (IL-1ab) was used to neutralize the activity of IL-1ß, mice received intravitreous injections of 500 ng IL-1ab or vehicle on P12 and P14, respectively, too. At P17, the mice were euthanized and their eyes were enucleated. The expression levels of IL-1ß, glutamine synthetase (GS) and l-glutamate/l-aspartate transporter (GLAST) in retinas with different treatments were detected. In addition, wild-type C57BL/6J mice received intravitreous injections of IL-1ß or PEDF. After 24 h, the expression of GS and GLAST in the retinas was also detected. Furthermore, high-performance liquid chromatography (HPLC) was performed to determine the glutamate concentrations in retinas with different treatments. RESULTS: The expression of IL-1ß and levels of glutamate were increased in retinas with OIR, while the expression of GS and GLAST was decreased. Administration of PEDF ameliorated the characteristic changes in retinas of OIR mice. And neutralization of IL-1ß by administration of IL-1ab increased GS and GLAST expression in retinas with OIR. Moreover, the effects of IL-1ß on GS and GLAST expression and unbalanced glutamate levels were inhibited after receiving intravitreous injections of PEDF in retinas of normal mice. CONCLUSIONS: These results suggested that PEDF might up-regulate GS and GLAST expression and decrease glutamate levels by suppressing the role of IL-1ß as an anti-inflammatory factor under hypoxia, and these functions may underlie the neuroprotective effects of PEDF.

Improvement and optimization of standards for a preclinical animal test model of laser induced choroidal neovascularization.

BACKGROUND: As the murine model of laser-induced choroidal neovascularization (CNV) is becoming the most established and commonly utilized model worldwide for studying the pathogenesis of CNV and its response to treatment, specific operating standards are yet to be clarified. The purpose of this study is to compare the lesion size of CNV in mice with different ages, sex, durations of CNV process, and treated positions of laser spots, to make recommendations that may improve and optimize the quality of the model. METHODS AND RESULTS: C57/BL6 mice of different ages were treated with diode laser photocoagulation per eye and perfused with PBS containing fluorescein-labeled dextran at different time of observation. Choroid flat mounts, were then examined by fluorescence microscopy for the measurement of CNV area. Messenger-RNA expression levels of several angiogenic cytokines in eye cups of male and female C57BL/6 mice at 5-8 and 16-20 week-old were analyzed by real-time RT-PCR assay. The results showed significantly more CNV area in eyes of female mice compared to male mice with the expression level of several angiogenic cytokines elevated. 16-20-week-old female mice developed the biggest area of CNV. The mean area of CNV increased significantly at the 14th day after photocoagulation. Laser spots delivered 1PD away from the optic disc induced the biggest area of CNV compared to those 2PD or 3PD away. Interaction of NV was observed in laser spots delivered less than 1PD away from each other. CONCLUSION: The current results suggest that 16-20-week-old female C57BL/6 mice developed the most distinct CNV lesion size with laser spots delivered 1PD away from the optic disc. The best time to observe and analyze is the 14th day after photocoagulation.