Decreased endostatin in db/db retinas is associated with optic disc intravitreal vascularization.

Endostatin, a naturally cleaved fragment of type XVIII collagen with antiangiogenic activity, has been involved in the regulation of neovascularization during diabetic retinopathy. Here, the intracellular distribution of endostatin in healthy mouse and human neuroretinas has been analyzed. In addition, to study the effect of experimental hyperglycemia on retinal endostatin, the db/db mouse model has been used. Endostatin protein expression in mouse and human retinas was studied by immunofluorescence and Western blot, and compared with db/db mice. Eye fundus angiography, histology, and immunofluorescence were used to visualize mouse retinal and intravitreal vessels. For the first time, our results revealed the presence of endostatin in neurons of mouse and human retinas. Endostatin was mainly expressed in bipolar cells and photoreceptors, in contrast to the optic disc, where endostatin expression was undetectable. Diabetic mice showed a reduction of endostatin in their retinas associated with the appearance of intravitreal vessels at the optic disc in 50% of db/db mice. Intravitreal vessels showed GFAP positive neuroglia sheath, basement membrane thickening by collagen IV deposition, and presence of MMP-2 and MMP-9 in the vascular wall. All together, these results point that decreased retinal endostatin during experimental diabetes is associated with optic disc intravitreal vascularization. Based on their phenotype, these intravitreal vessels could be neovessels. However, it cannot be ruled out the possibility that they may also represent persistent hyaloid vessels.

TIM2 modulates retinal iron levels and is involved in blood-retinal barrier breakdown.

Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and the progression of several retinopathies. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter protein, through its binding to Scara5 and TIM2 membrane receptors. In this study, the presence and iron-related functions of TIM2 in the mouse retina were investigated. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly in Müller cells. Experimental TIM2 downregulation in the mouse retina promoted, probably due to a compensatory mechanism, Scara5 overexpression that increased retinal ferritin uptake and induced iron overload. Consecutive reactive oxygen species (ROS) overproduction and vascular endothelial growth factor (VEGF) overexpression led to impaired paracellular and transcellular endothelial transport characterized by tight junction degradation and increased caveolae number. In consequence, blood-retinal barrier (BRB) breakdown and retinal edema were observed. Altogether, these results point to TIM2 as a new modulator of retinal iron homeostasis and as a potential target to counteract retinopathy.

Ischemic etiopathogenesis as the possible origin of post-double baloon enteroscopy pancreatitis. A porcine model study.

The aim is to evaluate the pancreatic vascular-ischemic effects related to double balloon enteroscopy in the porcine model as a possible etiopathogenesis of post-enteroscopic pancreatitis. For this reason we carry out two independent experiments in a porcine animal model. In the first arm protocol (group I), 10 animals underwent 90 minutes of oral enteroscopy with 7 days follow-up.The levels of amylase, lipase and C-reactive protein were measured at T0 basal-T1 -90 min, T2-24, T3-7 days. Also we perform upper gastrointestinal endoscopy in a control group. At 7 days, the animals of experimental protocol-I had their pancreases removed for a pathological and immunohistochemical study to evaluate vascular epithelial growth factor (VEGF) expression.The second experimental protocol in this study aims to evaluate possible changes in vascular topography due to the double balloon enteroscopy (DBE). Group-II (10 animals) underwent oral enteroscopy and selective angiography of the cranial mesenteric artery and celiac trunk. None of the group I or control group animals presented pancreatitis, although the biochemical results for group-I showed increases in the levels of amylase, lipase and C reactive protein at 24 hours. The microscopic study for group-I showed pancreatic necrotic foci and positive VEGF expression, though these changes were not expressed in the control group.These foci were found in 50% of the group I animals and in relation to the total of the parenchyma were quantified at 6% of the pancreas. The results for group-II showed that the enteroscopy caused mobilization of the mesenteric vascular axis, with signs of both intestinal and pancreatic hypoperfusion. The conclusions of this study are that, after enteroscopy in the porcine model, pancreatic necrotic foci are produced, in addition to ischemic phenomena causing VEGF expression. This could be related to episodes of visceral hypoperfusion caused by vascular alterations on a topographic level. This can be related to the possible ischemic etiopathogenesis described for post-enteroscopic pancreatitis.

Ferritin But Not Iron Increases in Retina Upon Systemic Iron Overload in Diabetic and Iron-Dextran Injected Mice.

Purpose: Iron overload causes oxidative damage in the retina, and it has been involved in the pathogeny of diabetic retinopathy, which is one of the leading causes of blindness in the adult population worldwide. However, how systemic iron enters the retina during diabetes and the role of blood retinal barrier (BRB) in this process remains unclear. Methods: The db/db mouse, a well-known model of type 2 diabetes, and a model of systemic iron overload induced by iron dextran intraperitoneal injection, were used. Perls staining and mass spectrophotometry were used to study iron content. Western blot and immunohistochemistry of iron handling proteins were performed to study systemic and retinal iron metabolism. BRB function was assessed by analyzing vascular leakage in fundus angiographies, whole retinas, and retinal sections and by studying the status of tight junctions using transmission electron microscopy and Western blot analysis. Results: Twenty-week-old db/db mice with systemic iron overload presented ferritin overexpression without iron increase in the retina and did not show any sign of BRB breakdown. These findings were also observed in iron dextran-injected mice. In those animals, after BRB breakdown induced by cryopexy, iron entered massively in the retina. Conclusions: Our results suggested that BRB protects the retina from excessive iron entry in early stages of diabetic retinopathy. Furthermore, ferritin overexpression before iron increase may prepare the retina for a potential BRB breakdown and iron entry from the systemic circulation.

Intercapillary bridging cells: immunocytochemical characteristics of cells that connect blood vessels in the retina.

Intervascular bridges are fibrous strands that connect neighboring capillaries. These strands present associated cells, intervascular bridging cells (IBCs), whose nature and functional significance remains controversial. The aim of this study was to characterize the immunophenotype of IBCs, and contribute to understand their mechanical and intercellular communication properties in the retina. Quantification and retinal distribution of IBCs were also determined. For this purpose, C57BL/6N and nestin-GFP transgenic mice, as well as human retinas, were used. Whole-mount retinas were studied by means of immunohistochemistry and cytochemistry, and isolation of retinal vasculature was achieved by trypsin/pepsin digest technique. PAS reaction and the immunolabeling with anti-collagen IV and laminin antibodies revealed that IBCs were completely surrounded by a basement membrane, connecting two or more neighboring capillaries. IBCs were scarce and their number decreased with age. They were preferentially localized in the deep vascular plexus. In a murine model of experimental glaucoma, methylcellulose injected eyes showed retinal neovascularization and increased number of IBCs in the deep vascular plexus. IBCs were marked with anti-NG2, anti-PDGFR-ß and anti-CD34 antibodies, and with tomato lectin, and were negative for PECAM-1. IBCs expressed nestin and filamentous actin, but desmin and α-smooth muscle actin were not detected. Moreover, these cells expressed the gap junction protein connexin 43. These results showed that IBCs had a pericytic nature since they expressed NG2 and the receptor for PDGF-B, and they were negative for PECAM-1. However, they were marked with CD34 and the tomato lectin, suggesting that they constitute a special subtype of pericytes, sharing characteristics with endothelial cells. IBCs presumably present mechanical functions due to the presence of filamentous actin. Connexin 43 was found in IBCs, suggesting that these cells allow intercellular communication between adjacent capillaries. This may represent an advantage for vasomotor tone integration and coordination in blood vessels without innervation, such as those of the retina.

Cold ischaemia, innate immunity and deterioration of the glomerular filtration barrier in antibody-mediated acute rejection.

BACKGROUND: In renal transplantation, cold ischaemia (CI) determines acute rejection through innate immunity among others. Acute rejection episodes are a risk factor for late allograft dysfunction and proteinuria. This implies some alteration of the glomerular filtration barrier (GFB). Besides its effects on acute rejection, we hypothesized that CI might somehow damage the GFB being directly responsible for late proteinuria. METHODS: On rat kidney allografts suffering from antibody-mediated acute rejection with or without CI and compared with syngeneic grafts, we quantified the gene expression of innate and adaptive immune mediators and assessed the capillary glomerular basement membranes (CapBM) by immunostaining collagen-IV (ColIV). ColIV was also assessed in equivalent groups from a previous chronic study followed up for 24 weeks. RESULTS: CI up-regulated enzymes critical in the stabilization of collagen chains, increasing ColIV deposition and thickening the CapBM. CI increased the C4d and IgG deposits within grafts, amplified innate immunity (heat shock protein 70, fibronectin, Toll-like-receptor-4 and MyD88) and synergized with alloreactivity in triggering adaptive response through CD40. CONCLUSIONS: Initial CI increased the ColIV deposition in CapBM, damaging the GFB and being responsible for part of the proteinuria associated with late allograft dysfunction. This deterioration of the GFB is related to the early innate immunity activation and subsequent up-regulation of CD40 in acute rejected grafts. In chronic rejected allografts, thickened CapBM may be a consequence of an unresolved immune-inflammatory response worsened by CI.

Increased intraocular insulin-like growth factor-I triggers blood-retinal barrier breakdown.

Blood-retinal barrier (BRB) breakdown is a key event in diabetic retinopathy and other ocular disorders that leads to increased retinal vascular permeability. This causes edema and tissue damage resulting in visual impairment. Insulin-like growth factor-I (IGF-I) is involved in these processes, although the relative contribution of increased systemic versus intraocular IGF-I remains controversial. Here, to elucidate the role of this factor in BRB breakdown, transgenic mice with either local or systemic elevations of IGF-I have been examined. High intraocular IGF-I, resulting from overexpression of IGF-I in the retina, increased IGF-I receptor content and signaling and led to accumulation of vascular endothelial growth factor. This was parallel to up-regulation of vascular Intercellular adhesion molecule I and retinal infiltration by bone marrow-derived microglial cells. These alterations resulted in increased vessel paracellular permeability to both low and high molecular weight compounds in IGF-I-overexpressing retinas and agreed with the loss of vascular tight junction integrity observed by electron microscopy and the altered junctional protein content. In contrast, mice with chronically elevated serum IGF-I did not show alterations in the retinal vasculature structure and permeability, indicating that circulating IGF-I cannot initiate BRB breakdown. Consistent with a key role of IGF-I signaling in retinal diseases, a strong up-regulation of the IGF-I receptor in human retinas with marked gliosis was also observed. Thus, this study demonstrates that intraocular IGF-I, but not systemic IGF-I, is sufficient to trigger processes leading to BRB breakdown and increased retinal vascular permeability. Therefore, therapeutic interventions designed to counteract local IGF-I effects may prove successful to prevent BRB disruption.

Endothelial cell transduction in primary cultures from regressing mesonephros.

Loss of renal function during normal aging is associated with vascular alterations. Consequently, new therapeutic approaches, including gene therapy, to protect renal endothelial cells are expected to be greatly beneficial. Quail mesonephros is a transitory embryonic kidney that has been used for the study of vascular development and involution. Vascular alterations in regressing mesonephros are similar to those observed in aging kidney. In the present study, we examined adenovirus-mediated gene transfer to endothelial cells in primary cultures from developing and regressing quail mesonephros. Quail embryos with developing and regressing mesonephros were examined on day 6 (30HH) and day 11 (40HH) of incubation, respectively. The senescence markers, associated beta-galactosidase activity and p16(INK4a), were examined in whole mesonephros. Quail embryos were injected intracardiacally with adenoviral vectors (rAd-CMV-LacZ) and endothelial cell transduction examined. In addition, primary cell cultures from mesonephros were exposed to adenoviral vectors. Endothelial cells in primary cultures were identified as QH1(+), LEP100(-) and acidic phosphatase(-) cells and adenovirus-transduced cells were those positive for bacterial-associated beta-galactosidase activity. We report that endothelial cells in the whole regressing mesonephros and primary cell cultures expressed senescence markers. In addition, we observed that adenoviral vectors were able to transduce endothelial cells in the whole regressing mesonephros, and that cultured endothelial and macrophagic cells from the regressing mesonephros were more efficiently transduced than those derived from the developing mesonephros. Our results suggest that quail mesonephros provides a practical model to assay gene transfer to endothelial cells in regressing/senescent vessels.

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.

Menstrual cycle in four New World primates: Poeppig's woolly monkey (Lagothrix poeppigii), red uakari (Cacajao calvus), large-headed capuchin (Sapajus macrocephalus) and nocturnal monkey (Aotus nancymaae).

Genital organs from 33 nocturnal monkeys Aotus namcymaae, 29 Poeppig's woolly monkeys (Lagothrix poeppigii), 21 red uakaris (Cacajao calvus) and 11 large-headed capuchins (Sapajus macrocephalus) were histologically analyzed in order to describe the endometrial changes related to the ovarian cycle. A. nancymaae and S. macrocephalus showed histological evidence of menstrual cycle with the detachment of the most superficial endometrium and the subepithelial reabsorption of the endometrial functional layer, explaining the extensive presence of both hemosiderin and fibrin clusters in the early follicular stages. In L. poeppigii, despite the presence of fibrin clusters promoting the remodeling of the endometrium, we did not observe the detachment of the functional layer of the endometrium, suggesting that this species presents a non-menstruating cycle. Finally, C. calvus showed no histological sign of menstrual phase. This reproductive information is useful to improve assisted reproductive techniques in non-human primates, and give us opportunity for comparative studies on the evolution of animal reproductive biology, including humans.

Vascular Interstitial Cells in Retinal Arteriolar Annuli Are Altered During Hypertension.

Purpose: It has been suggested that arteriolar annuli localized in retinal arterioles regulate retinal blood flow acting as sphincters. Here, the morphology and protein expression profile of arteriolar annuli have been analyzed under physiologic conditions in the retina of wild-type, ß-actin-Egfp, and Nestin-gfp transgenic mice. Additionally, to study the effect of hypertension, the KAP transgenic mouse has been used. Methods: Cellular architecture has been studied using digested whole mount retinas and transmission electron microscopy. The profile of protein expression has been analyzed on paraffin sections and whole mount retinas by immunofluorescence and histochemistry. Results: The ultrastructural analysis of arteriolar annuli showed a different cell population found between endothelial and muscle cells that matched most of the morphologic criteria established to define interstitial Cajal cells. The profile of protein expression of these vascular interstitial cells (VICs) was similar to that of interstitial Cajal cells and different from the endothelial and smooth muscle cells, because they expressed ß-actin, nestin, and CD44, but they did not express CD31 and α-SMA or scarcely express F-actin. Furthermore, VICs share with pericytes the expression of NG2 and platelet-derived growth factor receptor beta (PDGFR-ß). The high expression of Ano1 and high activity of nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase observed in VICs was diminished during hypertensive retinopathy suggesting that these cells might play a role on the motility of arteriolar annuli and that this function is altered during hypertension. Conclusions: A novel type of VICs has been described in the arteriolar annuli of mouse retina. Remarkably, these cells undergo important molecular modifications during hypertensive retinopathy and might thus be a therapeutic target against this disease.

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.

A new system to reduce formaldehyde levels improves safety conditions during gross veterinary anatomy learning.

Dissection is a very useful method of learning veterinary anatomy. However, formaldehyde, which is widely used to preserve cadavers, is an irritant, and it has recently been classified as a carcinogen. In 1997, the Instituto Nacional de Seguridad e Higiene en el Trabajo [National Institute of Workplace Security and Hygiene] found that the levels of formaldehyde in our dissection room were above the threshold limit values. Unfortunately, no optimal substitute for formaldehyde is currently available. Therefore, we designed a new ventilation system that combines slow propulsion of fresh air from above the dissection table and rapid aspiration of polluted air from the perimeter. Formaldehyde measurements performed in 2004, after the introduction of this new system into our dissection laboratory, showed a dramatic reduction (about tenfold, or 0.03 ppm). A suitable propelling/aspirating air system successfully reduces the concentration of formaldehyde in the dissection room, significantly improving safety conditions for students, instructors, and technical staff during gross anatomy learning.

Blood Vessel Basement Membrane Alterations in Human Retinal Microaneurysms During Aging.

Purpose: Microaneurysms, considered a hallmark of retinal vascular disease, are present in aged retinas. Here, the basement membrane of human retinal microaneurysms has been analyzed during aging. Methods: Retinas were obtained from 17 nondiabetic donors. Whole mount retinas and paraffin sections were marked immunohistochemically with antibodies against the main components of the blood basement membrane. Trypsin digestion and transmission electron microscopy also were performed. Results: Small microaneurysms presented increased expression of collagen IV, laminin, fibronectin, nidogen, and perlecan, along with basement membrane thickening. Unexpectedly, crosslinked fibrils of collagen III, a type of collagen absent in retinal capillaries, were found specifically in small microaneurysms. This was parallel to enhanced lysyl oxidase-like (LOXL) 2 and 4 expression. Large microaneurysms showed diminution of protein content, as well as disorganization, in their basement membrane. This was concomitant with an increased expression of matrix-metalloproteinase (MMP)-9 and plasminogen activator inhibitor (PAI)-1. Pericyte coverage declined between small and large microaneurysms. Conclusions: Thickening of the basement membrane in small microaneurysms by accumulation of matrix proteins probably produced by recruited pericytes, together with the appearance of crosslinked collagen III fibrils probably due to the action of LOXL2 and LOXL4, could be considered as compensatory mechanisms to strengthen the vascular wall in the early phase of microaneurysm formation. Later, increased activity of MMP-9 and PAI-I, which produce disruption of the blood basement membrane and expansion of microthrombi respectively, and loss of pericytes, which produces weakening of the vascular wall, could explain the wall dilation observed in the late phase of microaneurysm formation.

Cellular Senescence Is Associated With Human Retinal Microaneurysm Formation During Aging.

Purpose: Microaneurysms are present in healthy old-age human retinas. However, to date, no age-related pathogenic mechanism has been implicated in their formation. Here, cellular senescence, a hallmark of aging and several age-related diseases, has been analyzed in the old-age human retina and in the retina of a progeric mouse. Methods: Retinas were obtained from 17 nondiabetic donors and from mice deficient in Bmi1. Cellular senescence was analyzed by immunohistochemistry, senescent-associated ß-galactosidase activity assay, Sudan black B staining, conventional transmission electron microscopy, and immunoelectronmicroscopy. Results: Neurons, but not neuroglia, and blood vessels undergo cellular senescence in the old-age human retina. The canonical senescence markers p16, p53, and p21 were up-regulated and coexisted with apoptosis in old-age human microaneurysms. Senescent endothelial cells were discontinuously covered by fibronectin, and p16 colocalized with the ß1 subunit of fibronectin receptor α5ß1 integrin under the endothelial cellular membrane, suggesting anoikis as a mechanism involved in endothelial cell apoptosis. In a progeric mouse model deficient in Bmi1, where p21 was overexpressed, the retinal blood vessels displayed an aging phenotype characterized by enlarged caveolae and lipofuscin accumulation. Although mouse retina is not prone to develop microaneurysms, Bmi1-deficient mice presented abundant retinal microaneurysms. Conclusions: Together, these results uncover cellular senescence as a player during the formation of microaneurysms in old-age human retinas.

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

[This corrects the article DOI: 10.1371/journal.pone.0106974.].

Beta-cell death and mass in syngeneically transplanted islets exposed to short- and long-term hyperglycemia.

We studied the effects of hyperglycemia on beta-cell death and mass in syngeneically transplanted islets. Six groups of STZ-induced diabetic C57BL/6 mice were transplanted with 100 syngeneic islets, an insufficient beta-cell mass to restore normoglycemia. Groups 1, 2, and 3 remained hyperglycemic throughout the study. Groups 4, 5, and 6 were treated with insulin from day 7 before transplantation to day 10 after transplantation. After insulin discontinuation, group 6 mice achieved definitive normoglycemia. Grafts were harvested at 3 (groups 1 and 4), 10 (groups 2 and 5), and 30 (groups 3 and 6) days after transplantation. On day 3, the initially transplanted beta-cell mass (0.13 +/- 0.01 mg) was dramatically and similarly reduced in the hyperglycemic and insulin-treated groups (group 1: 0.048 +/- 0.002 mg; group 4: 0.046 +/- 0.007 mg; P < 0.001). Extensive islet necrosis (group 1: 30.7%; group 4: 26.8%) and increased beta-cell apoptosis (group 1: 0.30 +/- 0.05%; group 4: 0.42 +/- 0.07%) were found. On day 10, apoptosis remained increased in both hyperglycemic and insulin-treated mice (group 2: 0.44 +/- 0.09%; group 5: 0.48 +/- 0.08%) compared with normal pancreas (0.04 +/- 0.03%; P < 0.001). In contrast, on day 30, beta-cell apoptosis was increased in grafts exposed to sustained hyperglycemia (group 3: 0.37 +/- 0.03%) but not in normoglycemic mice (group 6: 0.12 +/- 0.02%); beta-cell mass was selectively reduced in islets exposed to hyperglycemia (group 3: 0.046 +/- 0.02 mg; group 6: 0.102 +/- 0.009 mg; P < 0.01). In summary, even in optimal conditions, approximately 60% of transplanted islet tissue was lost 3 days after syngeneic transplantation, and both apoptosis and necrosis contributed to beta-cell death. Increased apoptosis and reduced beta-cell mass were also found in islets exposed to chronic hyperglycemia, suggesting that sustained hyperglycemia increased apoptosis in transplanted beta-cells.

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.

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.

Carbohydrate characterization of quail primordial germ cells during migration and gonadal differentiation.

A selection of lectins were used to study changes in the distribution of sugar residues in primordial germ cells (PGCs) during gonadal colonization and differentiation. Although the cytochemical characteristics of PGCs have been described in the chick, little is known about such characteristics in other avian species of interest to experimental biology. Therefore, we studied embryos of Japanese quail (Coturnix coturnix japonica) by light and laser confocal scanning microscopy, using the QH1 antibody as a tool to identify PGCs in both sexes and at all stages. LEA, WGA and RCA-I bound to PGCs in a similar way to QH1. LEA was the best marker for all stages. The presence of acid phosphatase and the intense reaction of LEA, WGA, RCA-I and WFA at the cell surface were shown to be a useful tool in the study of the migratory PGCs of the quail. Quails were sexed histologically in younger embryos than in chick; results were confirmed by PCR. The lectin-binding pattern changed drastically in differentiated gonads. Cell surface reactivity was almost entirely lost. Quail differentiating oogonia showed a characteristic binding pattern to QH1 and to the lectins WGA, RCA-I and WFA. Binding was observed in intense spots in the Golgi complex, and there was a specific PNA reaction. These results suggest that some selective sugar binding sites on the PGCs play a significant role in their migration, colonization and maturation.