Fatty Acid Binding Protein 4 Deficiency Protects against Oxygen-Induced Retinopathy in Mice

Retinopathy of prematurity (ROP) is a leading cause of blindness in children worldwide due to increasing survival rates of premature infants. Initial suppression, followed by increased production of the retinal vascular endothelial growth factor-A (VEGF) expression are key events that trigger the pathological neovascularization in ROP. Fatty acid binding protein 4 (FABP4) is an intracellular lipid chaperone that is induced by VEGF in a subset of endothelial cells. FABP4 exhibits a pro-angiogenic function in cultured endothelial cells and in airway microvasculature, but whether it plays a role in modulation of retinal angiogenesis is not known. We hypothesized that FABP4 deficiency could ameliorate pathological retinal vascularization and investigated this hypothesis using a well-characterized mouse model of oxygen-induced retinopathy (OIR). We found that FABP4 was not expressed in retinal vessels, but was present in resident macrophages/microglial cells and endothelial cells of the hyaloid vasculature in the immature retina. While FABP4 expression was not required for normal development of retinal vessels, FABP4 expression was upregulated and localized to neovascular tufts in OIR. FABP4−/− mice demonstrated a significant decrease in neovessel formation as well as a significant improvement in physiological revascularization of the avascular retinal tissues. These alterations in retinal vasculature were accompanied by reduced endothelial cell proliferation, but no effect on apoptosis or macrophage/microglia recruitment. FABP4−/− OIR samples demonstrated decreased expression of genes involved in angiogenesis, such as Placental Growth Factor, and angiopoietin 2. Collectively, our findings suggest FABP4 as a potential target of pathologic retinal angiogenesis in proliferative retinopathies

Comparative global immune-related gene profiling of somatic cells, human pluripotent stem cells and their derivatives: implication for human lymphocyte proliferation.

Human pluripotent stem cells (hPSCs), including embryonic stem cells (ESCs) and induced PSCs (iPSCs), represent potentially unlimited cell sources for clinical applications. Previous studies have suggested that hPSCs may benefit from immune privilege and limited immunogenicity, as reflected by the reduced expression of major histocompatibility complex class-related molecules. Here we investigated the global immune-related gene expression profiles of human ESCs, hiPSCs and somatic cells and identified candidate immune-related genes that may alter their immunogenicity. The expression levels of global immune-related genes were determined by comparing undifferentiated and differentiated stem cells and three types of human somatic cells: dermal papilla cells, ovarian granulosa cells and foreskin fibroblast cells. We identified the differentially expressed genes CD24, GATA3, PROM1, THBS2, LY96, IFIT3, CXCR4, IL1R1, FGFR3, IDO1 and KDR, which overlapped with selected immune-related gene lists. In further analyses, mammalian target of rapamycin complex (mTOR) signaling was investigated in the differentiated stem cells following treatment with rapamycin and lentiviral transduction with specific short-hairpin RNAs. We found that the inhibition of mTOR signal pathways significantly downregulated the immunogenicity of differentiated stem cells. We also tested the immune responses induced in differentiated stem cells by mixed lymphocyte reactions. We found that CD24- and GATA3-deficient differentiated stem cells including neural lineage cells had limited abilities to activate human lymphocytes. By analyzing the transcriptome signature of immune-related genes, we observed a tendency of the hPSCs to differentiate toward an immune cell phenotype. Taken together, these data identify candidate immune-related genes that might constitute valuable targets for clinical applications

Oxidation of LDL lipids by different oxidants

Dottorato di ricerca in fisiopatologia sperimentale. 12. ciclo. Coordinatore Vanio Vannini Supervisore Aldo TomasiConsiglio Nazionale delle Ricerche - Biblioteca Centrale - P.le Aldo Moro, 7, Rome; Biblioteca Nazionale Centrale - P.za Cavalleggeri, 1, Florence / CNR - Consiglio Nazionale delle RichercheSIGLEITItal

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FABP4 expression and function during normal retinal development.

<p>(A) qPCR quantification of FABP4 mRNA in total retinas from P1 to P33 WT mice. Results are expressed as RE (relative expression) normalized to housekeeping genes (mean ± SEM, n = 3–4). FABP4 is significantly induced during the postnatal stage of retinal development. (B) FABP4 distribution was determined on retina flat-mounts and paraffin sections. At P7, FABP4 expression (red) was not observed in the inner retinal vessels stained by the pan-EC marker EMCN (green). FABP4 was strongly detected in the vitreal macrophages (arrowheads) and hyaloid endothelial cells (arrows). Paraffin sections of adult retina demonstrate faint, but uniform expression of FABP4 in all retinal layers. (C) WT and FABP4^−/− P7 flat-mount retinas were immunostained with Collagen IV to identify the vascular network. (D) Vascular coverage and density was similar in WT and FABP4^−/− animals indicating that FABP4 expression is not required for normal retinal vascularization. (E) Hematoxylin and eosin stained sagittal retinal sections of adult WT and FABP4^−/− mice showed no morphological anomalies or degeneration in FABP4^−/− animals. gcl: ganglion cell layer; ipl: inner plexiform layer; inl: inner nuclear layer; onl: outer nuclear layer; os: outer segment.</p

Real-time PCR primer sequences.

<p>Real-time PCR primer sequences.</p

Specific induction of FABP4 in neovessels during OIR.

<p>(A) qPCR quantification of FABP4 mRNA in total retinas from P12 to P17 in control (RA = room air) and OIR WT mice. Results are expressed as RE (relative expression) normalized to housekeeping genes (mean ± SEM, n = 3–4). FABP4 mRNA is significantly induced during the angiogenic phase of OIR. (B) Quantification of FABP4 protein expression in P17 RA control and OIR retinas by immunoblot and densitometry analysis (mean ± SEM, n  =  6) confirmed up-regulation of FABP4 expression during pathological neovascularization. (C) Quantification of circulating FABP4 demonstrated a significant decrease of serum FABP4 levels in OIR P17 WT mice compared to control room air (RA). Results are presented as mean ± SEM based on n = 3–4 individual animals. (D) FABP4 distribution was determined on P17 retina flat-mounts from OIR animals co-stained with the pan-endothelial marker BS1. FABP4 is expressed in neovascular tufts (arrowheads) and absent from adjacent normal blood vessels (arrows). Specificity of the FABP4 staining is confirmed using FABP4^−/− retina. Scale bar is 100 µm.</p

Protective effect of FABP4 deficiency in OIR is not associated with changes in macrophage/microglia recruitment.

<p>Activated and neovascular tuft-associated Iba1 positive (red) cells were quantified on FITC-BS1-stained (green) retina flat-mounts of P17 WT and FABP4^−/− OIR mice. There were no significant differences in the number of macrophages/microglia recruited to hypoxic retina or those associated with the neovessels (mean ± SEM, n = 9–10).</p