Marc Navarro

Increased ocular levels of IGF-1 in transgenic mice lead to diabetes-like eye disease

IGF-1 has been associated with the pathogenesis of diabetic retinopathy, although its role is not fully understood. Here we show that normoglycemic/normoinsulinemic transgenic mice overexpressing IGF-1 in the retina developed most alterations seen in human diabetic eye disease. A paracrine effect of IGF-1 in the retina initiated vascular alterations that progressed from nonproliferative to proliferative retinopathy and retinal detachment. Eyes from 2-month-old transgenic mice showed loss of pericytes and thickening of basement membrane of retinal capillaries. In mice 6 months and older, venule dilatation, intraretinal microvascular abnormalities, and neovascularization of the retina and vitreous cavity were observed. Neovascularization was consistent with increased IGF-1 induction of VEGF expression in retinal glial cells. In addition, IGF-1 accumulated in aqueous humor, which may have caused rubeosis iridis and subsequently adhesions between the cornea and iris that hampered aqueous humor drainage and led to neovascular glaucoma. Furthermore, all transgenic mice developed cataracts. These findings suggest a role of IGF-1 in the development of ocular complications in long-term diabetes. Thus, these transgenic mice may be used to study the mechanisms that lead to diabetes eye disease and constitute an appropriate model in which to assay new therapies.

Scavenger function of resident autofluorescent perivascular macrophages and their contribution to the maintenance of the blood-retinal barrier.

PURPOSE The retina contains two distinct populations of monocyte-derived cells: perivascular macrophages, and microglia. The present study was undertaken to evaluate the presence and function in mouse and human retinas of a subtype of resident perivascular macrophages with scavenger function, different from microglia, in physiological conditions and during retinopathy. METHODS Perivascular macrophages were characterized by means of confocal microscopy, electron microscopy, and flow cytometry analyses. Two murine models of blood-retinal barrier breakdown and photoreceptor degeneration were used to analyze the role of these macrophages during retinopathy. RESULTS The macrophages analyzed constituted a small population of resident perivascular cells different from microglia, since they were Iba-1 negative. Although these cells expressed F4/80 and CD11b antigens in common with microglia, they also expressed BM8 and MOMA-2 epitopes, which are macrophagic markers not expressed by microglia. Perivascular macrophages emitted autofluorescence due to cytoplasmic inclusions containing protein-bound oxidized lipids. They constitutively expressed the scavenger receptor class A and moved along blood vessels, providing an additional coating to thinner areas of the basement membrane. Moreover, they accumulated blood-borne horseradish peroxidase and acetylated low-density lipoprotein in healthy retinas. In addition, during blood-retinal barrier breakdown and photoreceptor degeneration, these cells migrated to the lesion site. CONCLUSIONS All these morphologic and functional features are consistent with those described for brain Mato cells. Thus, this study showed the presence of autofluorescent perivascular macrophages, different from microglia, with a scavenger function that may contribute to the maintenance of the blood-retinal barrier in healthy conditions and that are also involved in retinopathy.

Albendazole sulphoxide enantiomers in pregnant rats' embryo concentrations and developmental toxicity.

Three single oral doses (8.5, 10, and 14 mg/kg) of a racemic formulation of albendazole sulphoxide (ABZSO) were administered to pregnant rats on day 10 of gestation. Mother plasma and embryo concentrations of ABZSO enantiomers and albendazole sulphone (ABZSO(2)) were determined 9 h after administration. The (-)-ABZSO enantiomer showed higher peak concentrations in both maternal plasma and embryo than the (+) enantiomer. An increase in embryo concentrations of ABZSO enantiomers and ABZSO(2) was only observed when dose rose to 14 mg/kg. There was an increase in resorption when the dose increased, but significant differences were only found in the higher dose group when compared with the other groups. The incidence of external and skeletal malformations (mostly of the tail, vertebrae and ribs) rose significantly in the 10 mg/kg group, producing almost 20% and 90% of malformed fetuses, respectively, and gross external and skeletal abnormalities in the thoracic region and limbs were also found.

Sex differences in the disposition of albendazole metabolites in sheep.

Sex differences in the disposition of albendazole metabolites in sheep after oral administration of 20 mg/kg of netobimin have been studied. Some kinetic parameters of both metabolites show statistical differences between sexes; the sulphoxide and sulphone t1/2beta and MRT were lower in male animals than in females. Peak concentrations and AUC of sulphone metabolites were higher in males suggesting a greater oxidation rate compared with females. Urine excretion of albendazole metabolites, sulphoxide, sulphone, and amino sulphone appeared to be greater in female sheep than in males, mainly the sulphoxide metabolite. These differences between sexes can be caused by male sexual hormones, because testosterone and progesterone can induce or inhibit the microsomal Cytochrome P450 metabolism. Plasma protein-binding of albendazole sulphoxide and albendazole sulphone has been studied between male and female sheep, also their binding to sheep albumin and globulins. Both albendazole metabolites readily bind to sheep albumin and globulins. Male animals show a significantly lower binding of albendazole metabolites than females. These differences could be responsible for the non-esterified fatty acids (NEFA) present in the plasma. Males have significantly higher plasma levels of NEFA than females and which may compete with albumin for binding to albendazole metabolites.

Developmental toxicity in rat fetuses exposed to the benzimidazole netobimin

Netobimin (NTB) is a prodrug of albendazole (ABZ) and is used as a broad-spectrum anthelmintic both in human and veterinary medicine. Pregnant Sprague-Dawley rats were treated po with 50, 59.5 and 70.7 mg/kg of NTB on Gestational Day (GD) 10. The results, observed on GD 20, demonstrated that NTB induced a significant increase of resorptions. Moreover, decreased fetal body weight and an increase in skeletal malformations were observed in treated groups. We report the first study in which vascular malformations are described in rats after the administration of a benzimidazole compound. An interesting relationship between intercostal vessel and rib malformations was found.

The Quail Mesonephros: A New Model for Renal Senescence?

Background/Aims: Renal senescence during normal aging is associated with specific vascular alterations and tissue degeneration. Although the degenerative program executed during embryonic kidney development is known to include vascular alterations, studies yet have to examine whether it involves replicative senescence. In this study, we assessed the potential of the quail mesonephros, a transitory embryonic kidney, as a model of human renal senescence. Methods: Quail embryos with developing or degenerating mesonephros were studied on day 6 or day 11 of incubation, respectively. Senescence-associated β-galactosidase activity, a marker of replicative senescence, was examined on whole mounts and sections. Senescent vascular characterization was performed by the scanning electron-microscopic analysis of vascular corrosion casts. Results: Senescence-associated β-galactosidase activity was found only in old mesonephros. Moreover, at 11 days of incubation glomerular capillaries showed discontinuities and were thinner and more tortuous than those observed at 6 days, characteristics also reported for the aging human kidney. Conclusion: The degenerating quail mesonephros is a potential model of renal senescence, showing biochemical and morphological characteristics of the aging human kidney.

Microvascular assembly and cell invasion in chick mesonephros grafted onto chorioallantoic membrane

Embryonic tissues, in common with other tissues, including tumours, tend to develop a substantial vasculature when transplanted onto the chorioallantoic membrane (CAM). Studies conducted to date have not examined in any detail the identity of vessels that supply these grafts, although it is known that the survival of transplanted tissues depends on their ability to connect with CAM vessels supplying oxygen and nutrients. We grafted the mesonephros, a challenging model for studies in vascular development, when it was fully developed (HH35). We used reciprocal chick‐quail transplantations in order to study the arterial and venous connections and to analyse the cell invasion from the CAM to the organ, whose degeneration in normal conditions is rapid. The revascularization of the grafted mesonephros was produced by the formation of peripheral anastomoses between the graft and previous host vasculatures. The assembly of graft and CAM blood vessels occurred between relatively large arteries or veins, resulting in chimeric vessels of varying morphology depending on their arterial or venous status. Grafts showed an increased angiogenesis from their original vasculature, suggesting that the normal vascular degeneration of the mesonephros was partially inhibited. Three types of isolated host haemangioblast were identified in the mesonephros: migrating angioblast‐like cells, indicating vasculogenesis, undifferentiated haematopoietic cells and macrophages, which might have been involved in the angiogenesis. Tomato lectin was found to bind activated macrophages in avian embryos.

Morphological characterization of pecteneal hyalocytes in the developing quail retina

The periphery of the vitreous body contains a population of cells termed hyalocytes. Despite the existence for more than one century of publications devoted to the pecten oculi, a convoluted coil of blood vessels that seems to be the primary source of nutrients for the avian avascular retina, little information can be found concerning the pecteneal hyalocytes. These cells are situated on the inner limiting membrane in close relationship with the convolute blood vessels. To characterize the origin and macrophagic activity of pecteneal hyalocytes, we have analysed two different stages of quail eye development using histochemistry and immunohistochemistry. Pecteneal hyalocytes express the QH1 epitope and cKit, confirming that these cells belong to the haematopoietic system. They also express vimentin, an intermediate filament protein present in cells of mesenchymal origin and very important for differentiation of fully active macrophages. However, similarly as described in porcine hyalocytes, pecteneal hyalocytes express the glial fibrillary acidic protein, a recognized neuroglial marker. Pecteneal hyalocytes did not express other neuroglial markers, such as glutamine synthetase or S100. Acidic phosphatase was activated and Lep100 was found in secondary lysosomes, confirming phagocytic activity of pecteneal hyalocytes during ocular development. Pecteneal hyalocytes strongly react with RCA‐I, WFA, WGA, PNA, SNA, LEA and SBA lectins, whereas other avian macrophages from thymus and the bursa of Fabricius did not bind PNA, SNA and LEA lectins. Interestingly, WGA lectin reacts with all kinds of avian macrophages, including pecteneal hyalocytes, probably reflecting the specific binding of WGA to components of the phagocytic and endocytic pathways. In conclusion, pecteneal hyalocytes are a special subtype of blood‐borne macrophages that express markers not specifically associated with the haematopoietic system.

Morphogenesis of Blood Vessels in the Head Muscles of Avian Embryo: Spatial, Temporal, and VEGF Expression Analyses

Adult skeletal muscle is a highly vascularized tissue, but the development of intramuscular endothelial networks has not been well studied. In quail embryos, QH1‐positive angioblasts are present and moving throughout myogenic head mesoderm before the onset of primary myotube formation. On day 5 of incubation, concurrent with early myotube formation and aggregation, angioblasts establish a transient vascular plexus surrounding the myogenic condensations. Between days 5 and 9, the intramuscular vessels form an irregular network of endothelial cords and patent channels and only later are the parallel arrays of capillaries characteristic of adult muscles established. Microinjections using India ink, QH1, and Mercox resin reveal that these intramuscular capillaries are typically not connected to systemic vessels of the head until day 10, which is near the end of primary myogenesis and corresponds to the onset of muscular function. Morphometric analyses performed during primary myogenesis stages show a decrease in muscle cell density but no significant changes in intramuscular vascular density between days 5 and 9. This finding was surprising, as it is generally assumed that muscle growth requires elevated oxygen and nutrient levels. Moreover, there are no significant morphometric differences in vascular supply to embryonic fast and slow muscles. Endothelial tissue density is similar in slow muscles (oculorotatory, e.g., lateral rectus), fast muscles (mandibular depressor), and mixed muscles, in which the fiber types can be interspersed (jaw adductors) or segregated (branchiomandibular). Vascular endothelial growth factor (VEGF) protein is abundant in myotubes but not endothelial cells within both fast and slow head muscles at days 7 and 9. However, in some mixed muscles, only a minority of myotubes, which do not correspond to one specific fiber type, express VEGF. These results document a dynamic set of intramuscular and perimuscular angiogenic reorganizations during avian head myogenesis. Thus far, no vasculogenic distinctions between fast and slow muscles have been observed, although muscle heterogeneity in VEGF expression is evident. Developmental Dynamics 227:470–483, 2003.

L-Ferritin Binding to Scara5: A New Iron Traffic Pathway Potentially Implicated in Retinopathy

Iron is essential in the retina because the heme-containing enzyme guanylate cyclase modulates phototransduction in rods and cones. Transferrin endocytosis is the classical pathway for obtaining iron from the blood circulation in the retina. However, the iron storage protein ferritin has been also recently proposed as an iron carrier. In this study, the presence of Scara5 and its binding to L-ferritin was investigated in the retina. Our results showed that Scara5, the specific receptor for L-ferritin, was expressed in mouse and human retinas in many cell types, including endothelial cells. Furthermore, we showed that intravenously injected ferritin crossed the blood retinal barrier through L-ferritin binding to Scara5 in endothelial cells. Thus, suggesting the existence of a new pathway for iron delivery and trafficking in the retina. In a murine model of photoreceptor degeneration, Scara5 was downregulated, pointing out this receptor as a potential player implicated in retinopathy and also as a possible therapeutic target.